Screening methods for parp modulators

ABSTRACT

The present disclosure is related to methods of identifying Poly(ADP-ribose) polymerases (PARP) inhibitors, and the methods of using PARP probes.

CLAIM OF PRIORITY

This application is a divisional application of U.S. patent applicationSer. No. 16/397,410, filed on Apr. 29, 2019, which claims priority toU.S. Provisional Patent Application Ser. No. 62/664,595, filed on Apr.30, 2018, the entire contents of which are hereby incorporated byreference.

SEQUENCE LISTING

This application contains a Sequence Listing that has been submittedelectronically as an ASCII text file named SequenceListing.txt. TheASCII text file, created on Mar. 17, 2022, is 126 kilobytes in size. Thematerial in the ASCII text file is hereby incorporated by reference inits entirety.

TECHNICAL FIELD

The present disclosure is related to methods of identifyingPoly(ADP-ribose) polymerase (PARP) inhibitors.

BACKGROUND

Poly(ADP-ribose) polymerases (PARPs) are a family of 17 enzymes that cantransfer ADP-ribose from nicotinamide adenine dinucleotide (NAD⁺) toprotein and nucleic acid substrates¹⁻². The PARP enzymes family iscomprised of two subfamilies, monoPARPs and polyPARPs. MonoPARP enzymescatalyze the transfer of a single ADP-ribose group to a target aminoacid, while polyPARP enzymes catalyze the transfer of multipleADP-ribose groups to form polymers. While approved drugs that target thepolyPARPs exist, there are no potent and selective inhibitors of themonoPARPs. MonoPARPs are important regulators of the immune response³⁻⁴and are implicated in human diseases such as inflammation⁵ andcancer⁶⁻⁷, therefore small molecules that modulate the enzymaticactivity of monoPARPs can be useful therapeutics. Despite interest inthe development of monoPARP inhibitors, the field is lacking effectivehigh-throughput assays that can be used to screen for and characterizemodulators of monoPARP function. Thus, there is an urgent need forhigh-throughput assays that can be used to screen monoPARP modulators.

SUMMARY

The present disclosure is related to methods of identifying PARPmodulators.

The present invention is directed to a method of identifying aninhibitor for PARPs, the method comprising:

combining (i) a polypeptide comprising a PARP catalytic domain whereinthe polypeptide is labeled with a donor fluorophore, (ii) a PARP probe,wherein the PARP probe is labeled with an acceptor fluorophore, and(iii) a test compound;

exposing the donor fluorophore to excitation light;

measuring a signal produced by the acceptor fluorophore; and

identifying the test compound as an inhibitor for PARP based on thesignal produced by the acceptor fluorophore.

The present invention is further directed to a method of identifying aninhibitor for PARPs, the method comprising:

combining (i) a polypeptide comprising a PARP catalytic domain, whereinthe polypeptide is labeled with an acceptor fluorophore; (ii) a PARPprobe, wherein the PARP probe is labeled with a donor fluorophore, and(iii) a test compound;

exposing the donor fluorophore to excitation light;

measuring a signal produced by the acceptor fluorophore in the presenceof a test compound; and

identifying the test compound as an inhibitor for PARP based on thesignal produced by the acceptor fluorophore.

The present invention is further directed to a method of identifying aninhibitor for PARPs, the method comprising:

contacting a polypeptide comprising a PARP catalytic domain with a PARPprobe in the presence of a test compound, wherein the PARP probecomprises a fluorophore;

exposing the probe to polarized excitation light, thereby generatingfluorescence;

determining a fluorescence polarization value of the fluorescence; and

identifying the test compound as an inhibitor for PARP based on thefluorescence polarization value of the fluorescence.

The present invention is further directed to a method of identifying aninhibitor for PARPs, the method comprising:

contacting a fusion polypeptide with a PARP probe that comprises afluorophore, wherein the fusion polypeptide comprises a PARP catalyticdomain and a luciferase enzyme;

contacting the luciferase enzyme with a substrate to produce light,wherein the light can excite the fluorophore;

measuring a signal produced by the fluorophore in the presence of a testcompound; and

identifying the test compound as an inhibitor for PARP based on thesignal produced by the fluorophore.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Methods and materials aredescribed herein for use in the present invention; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control.

Other features and advantages of the invention will be apparent from thefollowing detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 is a schematic diagram illustrating an example of in vitroTR-FRET probe displacement assay. An inhibitor will outcompete thebiotinylated probe and decrease TR-FRET signal.

FIG. 2 is a schematic diagram illustrating an example of in vitrofluorescence polarization probe displacement assay. An inhibitor willoutcompete the fluorophore-labeled probe and decrease fluorescencepolarization signal.

FIG. 3 is a schematic diagram illustrating an example of bioluminescenceresonance energy transfer probe displacement assay. An inhibitor willoutcompete the fluorophore-labeled probe and decrease NanoBRET signal.

FIGS. 4A-4F. Validation results of the in vitro probe displacementbinding assays. Dose response curves for Compound A were generated usingeach assay to confirm that Probe C was outcompeted from the monoPARPenzyme. IC₅₀ values for Compound A were 7 nM (TIPARP), 80 nM (PARP10),200 nM (PARP12), 50 nM (PARP14), 60 nM (PARP15) and 100 nM (PARP16).

FIGS. 5A-5D. Validation results of the bioluminescence resonance energytransfer probe displacement binding assays. Dose response curves forcontrol compounds were generated using each assay to confirm that ProbeA was outcompeted from the monoPARP enzyme. IC₅₀ values for catalyticTIPARP Compound B=7 nM, full-length TIPARP Compound B=4 nM, PARP10Compound C=4 nM, PARP12 Compound B=170 nM, PARP14 Compound A=30 nM.

FIGS. 6A-6F show the amino acid sequences of PARPs.

DETAILED DESCRIPTION

PARPs refers to a family of proteins involved in a number of cellularprocesses such as DNA repair, genomic stability, and programmed celldeath.

The primary function of PARPs is to post-translationally modify targetproteins with ADP-ribose using NAD⁺ as substrate. The four best-studiedfamily members, PARP1 and PARP5a along with their close functionalhomologs PARP2 and PARP5b respectively, all generate poly(ADP-ribose)(PAR). The main role of these PARPs is to detect and initiate animmediate cellular response to metabolic, chemical, or radiation-inducedsingle-strand DNA breaks (SSB) by signaling the enzymatic machineryinvolved in the SSB repair. Once PARP detects a SSB, it binds to theDNA, undergoes a structural change, and begins the synthesis of apolymeric adenosine diphosphate ribose (poly (ADP-ribose)) chain, whichacts as a signal for the other DNA-repairing enzymes.

However, many other PARPs do not generate PAR, and instead attachADP-ribose as a monomer ADP-ribose (MAR) onto target proteins. Recentdata has shown that many of these MAR-generating PARPs (monoPARPs) canhave cancer relevant functions or inflammation related functions.MonoPARP enzymes have structurally related active sites which bind tonicotinamide adenine dinucleotide (NAD⁺) and catalyze the transfer ofadenosine diphosphoribose to a substrate amino acid. Enzyme-linkedimmunosorbent assays (ELISA) measuring the incorporation of biotin-NAD⁺to histones or to the monoPARP itself in an automodification reactionhave been used to screen for monoPARP modulators⁸. These reactions arenot catalytically efficient, and high concentrations of enzyme areneeded to generate sufficient turnover to be detected. Since the lowestmeasurable IC₅₀ in an enzyme assay is half of the total enzymeconcentration⁹, these assays are usually unable to differentiate andrank order very potent compounds. Thermal shift assays (TSA) have alsobeen used to screen inhibitors of monoPARPs¹⁰, however these assaysconsume large amounts of protein and are at best semi-quantitative sincecompounds with similar binding affinities can have different effects onprotein stabilization¹¹.

The present disclosure provides a more effective way to identify PARPmodulators, and provides a series of high affinity active site probesthat bind in the NAD⁺ pocket of monoPARPs which can be used to develophigh-throughput biophysical assays.

Poly (ADP-Ribose) Polymerases

There are 17 PARPs. The enzymatic activity and cancer relevant functionsof these PARPs are summarized in Table 1 below.

TABLE 1 Cancer Other NCBI Accession Related Cancers to PARP NamesActivity No. Catalytic Domain Functions target PARP1 ARTD1 PARNP_001609.2 Amino acids 788- DNA Repair, HR deficient (SEQ ID NO: 13)1014 of accession ERK/ NF-kB Elevated no. NP_001609.2 signaling, HeatERK/NF-kB shock signaling response PARP2 ARTD2 PAR NP_005475.2 Aminoacids 356- DNA Repair HR deficient (SEQ ID NO: 14) 583 of accession no.NP_005475.2 PARP3 ARTD3 MAR NP_001003931.3 Amino acids 313- DNA RepairDNA repair (SEQ ID NO: 15) 533 of accession deficient no. NP_001003931.3PARP4 vPARP MAR NP_006428.2 Amino acids 369- ARTD4 (SEQ ID NO: 16) 573of accession no. NP_006428.2 PARP5a TNKS1 PAR NP_003738.2 Amino acids1112- Telomere Elevated Wnt ARTD5 (SEQ ID NO: 17) 1317 of accessionMaintenance, Signaling no. NP_003738.2 Wnt Signaling, TelomeraseProteasome Dependent Regulation, Stress Granule Stress Granule PositiveSolid Assembly, Cell Tumors Division PARP5b TNKS2 PAR NP_079511.1 Aminoacids 959- Telomere Elevated Wnt ARTD6 (SEQ ID NO: 18) 1164 of accessionMaintenance, Signaling, no. NP_079511.1 Wnt Signaling TelomeraseDependent PARP6 ARTD17 MAR NP_001310451.1 Amino acids 394- NegativePotential (SEQ ID NO: 19) 620 of accession Regulator of Tumor no.Proliferation Suppressive NP_001310451.1 Functions TIPARP PARP7 MARNP_056323.2 Amino acids 449- ARTD14 (SEQ ID NO: 1) 657 of accession no.NP_056323.2 PARP8 ARTD16 MAR AAH37386.1 Amino acids 328- (SEQ ID NO: 20)494 of accession no. AAH37386.1 PARP9 BAL1 MAR AAH39580.1 Amino acids628- Cell Migration Metastatic ARTD9 (SEQ ID NO: 21) 850 of accessionCancers no. AAH39580.1 PARP10 ARTD10 MAR NP_116178.2 Amino acids 806-Inhibits Potential (SEQ ID NO: 2) 1025 of accession Myc and NF- Tumorno. NP_116178.2 kB signaling Suppressive Pro-apoptotic Functions PARP11ARTD11 MAR Q9NR21.2 Amino acids 123- (SEQ ID NO: 22) 338 of accessionno. Q9NR21.2 PARP12 ARTD12 MAR NP_073587.1 Amino acids 484- StressGranule Stress Granule (SEQ ID NO: 3) 698 of accession Assembly PositiveSolid no. NP_073587.1 Tumors PARP13 ZAP MAR NP_064504.2 Amino acids 716-Stress Granule Stress Granule ZC3HAV1 (SEQ ID NO: 23) 902 of accessionAssembly, Positive Solid ARTD13 no. NP_064504.2 miRNA-RISC Tumorsregulation PARP14 BAL2 MAR NP_060024.2 Amino acids 1605- B cellsurvival, Hematopoeitic ARTD8 (SEQ ID NO: 4) 1801 of accession CellMigration, malignancies, no. NP_060024.2 Stress Granule MetastaticAssembly Cancers PARP15 BAL3 MAR NP_689828.1 Amino acids 482- StressGranule Stress Granule ARTD7 (SEQ ID NO: 5) 678 of accession AssemblyPositive Solid no. NP_689828.1 Tumors PARP16 ARTD15 MAR NP_060321.3Amino acids 94- ER Unfolded UPR (SEQ ID NO: 6) 279 of accession Proteindependent no. NP_060321.3 Response

Among these enzymes, PARP1, PARP2, PARP5a and PARP5b can generate poly(ADP-ribose) (PAR). PARP3, PARP4, PARP6, TIPARP, PARP8, PARP9, PARP10,PARP11, PARP12, PARP13, PARP14, PARP15, and PARP16 are monoPARPs.

PARPs have multiple diverse functions in physiological pathwaysincluding cell migration, transcriptional regulation, signaltransduction, miRNA-mediated gene silencing, regulation of membraneorganelles and telomere length regulation. Additionally, PARPs functionin stress-responsive cellular pathways upon DNA damage, cytoplasmicstress, environmental stress and ER stress, activating DNA damagerepair, stress granule assembly, the heat shock response and the ERunfolded protein response pathways in response. Many of thesephysiological and stress response pathways are misregulated in cancer orinflammation.

Thus, the inhibitors of PARPs (e.g., monoPARPs) can have various uses.For example, they can be used to modulate (e.g., inhibit or facilitate)cell migration, transcriptional regulation, signal transduction, andgene silencing. They can also be used to modulate (e.g., inhibit orfacilitate) stress-responsive cellular pathways (e.g., upon DNA damage),or DNA damage repair pathways. In some embodiments, PARP inhibitors(e.g., monoPARP inhibitors) can be used to treat a disorder associatedwith PARP overexpression or overactivity. In some embodiments, PARPinhibitors (e.g., monoPARP inhibitors) can be used to treat cancers orinflammation.

A detailed description of PARPs and their functions can be found, e.g.,in Vyas et al., “New PARP targets for cancer therapy,” Nature ReviewsCancer 14.7 (2014): 502, which is incorporated herein by reference inits entirety.

The present disclosure provides methods of identifying PARP modulators,and also provides polypeptides (e.g., fusion polypeptides) comprising acatalytic domain of PARPs (e.g., PARP1, PARP2, PARP3, PARP4, PARP5a,PARP5b, PARP6, TIPARP, PARP8, PARP9, PARP10, PARP11, PARP12, PARP13,PARP14, PARP15, or PARP16). These polypeptides can be used in variousassays for identifying the modulators of interest (e.g., PARPinhibitors). As used herein, the term “catalytic domain” refers to aportion of an enzyme that has a catalytic activity. The catalytic domaincomprises the region of an enzyme that interacts with its substrate tocause the enzymatic reaction. In many cases, the active site is locatedin the catalytic domain, and the substrate binds to active site.

In some embodiments, the catalytic domain is the catalytic domain ofTIPARP (e.g., residues 449-657 of NP_056323.2 (SEQ ID NO: 1)), thecatalytic domain of PARP10 (e.g., residues 806-1025 of NP_116178.2 (SEQID NO: 2)), the catalytic domain of PARP12 (e.g., residues 484-698 ofNP_073587.1 (SEQ ID NO: 3)), the catalytic domain of PARP14 (e.g.,residues 1605-1801 of NP_060024.2 (SEQ ID NO: 4)), the catalytic domainof PARP15 (e.g., residues 482-678 of NP_689828.1 (SEQ ID NO: 5)), or thecatalytic domain of PARP16 (e.g., residues 5-279 of NP_060321.3 (SEQ IDNO: 6)).

In some embodiments, the catalytic domain is the catalytic domain ofPARP1 (e.g., residues 788-1014 of NP_001609.2 (SEQ ID NO: 13)), thecatalytic domain of PARP2 (e.g., residues 356-583 of NP_005475.2 (SEQ IDNO: 14)), the catalytic domain of PARP3 (e.g., residues 313-533 ofNP_001003931.3 (SEQ ID NO: 15)), the catalytic domain of PARP4 (e.g.,residues 369-573 of NP_006428.2 (SEQ ID NO: 16)), the catalytic domainof PARP5a (e.g., residues 1112-1317 of NP_003738.2 (SEQ ID NO: 17)), thecatalytic domain of PARP5b (e.g., residues 959-1164 of NP_079511.1 (SEQID NO: 18)), the catalytic domain of PARP6 (e.g., residues 394-620 ofNP_001310451.1 (SEQ ID NO: 19)), the catalytic domain of PARP8 (e.g.,residues 328-494 of AAH37386.1 (SEQ ID NO: 20)), the catalytic domain ofPARP9 (e.g., residues 628-850 of AAH39580.1 (SEQ ID NO: 21)), thecatalytic domain of PARP11 (e.g., residues 123-338 of Q9NR21.2 (SEQ IDNO: 22)), or the catalytic domain of PARP13 (e.g., residues 716-902 ofNP_064504.2 (SEQ ID NO: 23)).

In some embodiments, the polypeptide comprises a catalytic domain thathas a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% identical to the sequence of a catalytic domain asdescribed herein.

In some embodiments, the polypeptide (e.g., fusion polypeptide)comprises a region of a PARP, wherein the substrate binding pocket islocated in this region of the PARP. In some embodiments, the polypeptidecomprises residues 456-657 of NP_056323.2 (SEQ ID NO: 1), residues808-1025 of NP_116178.2 (SEQ ID NO: 2), residues 489-684 of NP_073587.1(SEQ ID NO: 3), residues 1611-1801 of NP_060024.2 (SEQ ID NO: 4),residues 481-678 of NP_689828.1 (SEQ ID NO: 5), or residues 5-279 ofNP_060321.3 (SEQ ID NO: 6).

In some embodiments, the polypeptide can be linked with a label (e.g., afluorophore). As used herein, the term “linked” refers to beingcovalently or non-covalently associated, e.g., by a chemical bond (e.g.,a peptide bond, or a carbon-carbon bond), by hydrophobic interaction, byVan der Waals interaction, and/or by electrostatic interaction.

The label can be a chemical or composition detectable by spectroscopic,photochemical, biochemical, immunochemical, chemical, or other physicalmeans. For example, useful labels include fluorescent dyes(fluorophores), luminescent agents, electron-dense reagents, enzymes(e.g., as commonly used in an ELISA, or luciferase), biotin, enzymesacting on a substrate (e.g., horseradish peroxidase), digoxigenin, ³²Pand other isotopes, haptens, and proteins which can be made detectable,e.g., by incorporating a radiolabel into the peptide or used to detectantibodies specifically reactive with the peptide. The term includescombinations of single labeling agents, e.g., a combination offluorophores that provides a unique detectable signature, e.g., at aparticular wavelength or combination of wavelengths. Any method known inthe art for conjugating label to a desired agent may be employed.

In some embodiments, the polypeptide can have a fusion tag (e.g., SEQ IDNO: 7, 8, 9, or 10). These fusion tags can be used to purifypolypeptides.

In some embodiments, the polypeptide can have an epitope (e.g., 6×His)that can be specifically recognized by an antibody (e.g., anti-6×Hisantibody). A label (e.g., fluorophore) can be conjugated to theantibody, thereby associating with the polypeptide.

In some embodiments, the polypeptide is a fusion polypeptide and cancomprise a luciferase (e.g., SEQ ID NO: 11). The luciferase can belocated at the N-terminus or the C-terminus of the polypeptide.

The disclosure also provides a nucleic acid sequence that is at least1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% identical to any nucleotide sequence asdescribed herein, and an amino acid sequence that is at least 1%, 2%,3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% identical to any amino acid sequence as described herein.In some embodiments, the nucleic acid sequence is at least 60%, 65%,70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to any nucleotide sequence as described herein. In someembodiments, the amino acid sequence is at least 60%, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical toany amino acid sequence as described herein.

To determine the percent identity of two amino acid sequences, or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes).The length of a reference sequence aligned for comparison purposes is atleast 80% of the length of the reference sequence, and in someembodiments is at least 90%, 95%, or 100%. The amino acid residues ornucleotides at corresponding amino acid positions or nucleotidepositions are then compared. When a position in the first sequence isoccupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position (as used herein amino acid or nucleic acid“identity” is equivalent to amino acid or nucleic acid “homology”). Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences, taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences. For purposes of the presentdisclosure, the comparison of sequences and determination of percentidentity between two sequences can be accomplished using a Blossum 62scoring matrix with a gap penalty of 12, a gap extend penalty of 4, anda frameshift gap penalty of 5.

In some embodiments, the disclosure relates to nucleotide sequencesencoding any peptides that are described herein, or any amino acidsequences that are encoded by any nucleotide sequences as describedherein. In some embodiments, the nucleic acid sequence is less than 10,20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 150, 200, 250, 300,350, 400, 500, 600, 700, 800, 900, or 1000 nucleotides. In someembodiments, the amino acid sequence is less than 5, 6, 7, 8, 9, 10, 20,30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180,190, 200, 400, 500, 600, 700, 800, 900, or 1000 amino acid residues.

PARP Probes

The present disclosure provides high affinity active site probes thatbind in the NAD⁺ pocket of PARPs (e.g., monoPARPs) which can be used todevelop high-throughput biophysical assays. These probes are based onthe structure-activity relationship (SAR) and binding mode of inhibitorsthat bind to the NAD⁺ pocket of monoPARP enzymes. Thus, as used herein,the term “PARP probe” refers to an agent (e.g., a small molecule) thatcan bind to the active site of a PARP.

Many enzyme inhibitors inhibit the functions of enzymes by preventing asubstrate from entering the enzyme's active site. Thus, if a testcompound can prevent an active site probe from binding to the enzyme'sactive site, the test compound can also prevent a substrate fromentering the enzyme's active site, thus working as an inhibitor.Therefore, in some embodiments, these PARP inhibitors can compete withPARP probes, bind to or occupy enzyme's active site, and/or displace thePARP probes.

In some embodiments, the PARP probe has a structure according to Formula(I):

or a salt thereof,wherein:

L is a linking group having 10-30 spacer atoms selected from C, N, O,and S connecting the N atom of the piperidinyl group of Formula (I) withgroup A; and

A is a fluorophore or an affinity tag.

In some embodiments,

L is:

wherein:a is 0, 1, or 2;b is 1-26; andc is 0, 1, or 2;wherein the sum of a+b+c is 1 to 26, orL is a chain of 5-30 atoms in length comprising —(CH₂CH₂O)_(d)— whereind is 2-10.

In some embodiments, A is an organic dye.

In some embodiments, A is biotin.

In some embodiments, A is:

or a salt thereof.

As used herein, the term “fluorophore” refers to a chemical compoundthat can re-emit light upon light excitation. The donor is thefluorophore that emits light of shorter wavelength which is used toexcite the acceptor, causing it to emit light of longer wavelength.Fluorophores can be organic molecules like fluorescein and similarorganic dye moieties. Fluorophores can also include inorganic componentslike transition metals.

An affinity tag is a chemical or polypeptide group that can bind toother chemical or polypeptide molecules covalently or non-covalently(e.g., preferably with high affinity). Examples of non-covalent affinitytags are biotin which binds to streptavidin and antibodies,hexahistidine which binds to nickel-nitrilotriacetic acid andantibodies, glutathione which binds to glutathione S-transferase andantibodies, etc. Examples of covalent affinity tags are primary aminessuch as lysine which react with N-hydroxysuccinamide, as well as freethiols such as cysteine which react with other free thiols to formdisulfide bonds. In some embodiments, the affinity tag is biotin.

Where a PARP probe “is labeled” with a fluorophore, the PARP probe cancontain an affinity tag that covalently or non-covalently binds with amolecule (e.g., streptavidin or an antibody) having a fluorophore. Wherea PARP probe comprises a fluorophore or an affinity tag, the fluorophoreor affinity tag is generally understood to be part of the probemolecule.

The PARP probes of the invention, including salts thereof, can beprepared using known organic synthesis techniques and can be synthesizedaccording to any of numerous possible synthetic routes.

The reactions for preparing compounds as described herein can be carriedout in suitable solvents which can be readily selected by one of skillin the art of organic synthesis. Suitable solvents can be substantiallynonreactive with the starting materials (reactants), the intermediates,or products at the temperatures at which the reactions are carried out,e.g., temperatures which can range from the solvent's freezingtemperature to the solvent's boiling temperature. A given reaction canbe carried out in one solvent or a mixture of more than one solvent.Depending on the particular reaction step, suitable solvents for aparticular reaction step can be selected by the skilled artisan.

Preparation of compounds as described herein can involve the protectionand deprotection of various chemical groups. The need for protection anddeprotection, and the selection of appropriate protecting groups, can bereadily determined by one skilled in the art. The chemistry ofprotecting groups can be found, for example, in T. W. Greene and P. G.M. Wuts, Protective Groups in Organic Synthesis, 3rd. Ed., Wiley & Sons,Inc., New York (1999), which is incorporated herein by reference in itsentirety.

Reactions can be monitored according to any suitable method known in theart. For example, product formation can be monitored by spectroscopicmeans, such as nuclear magnetic resonance spectroscopy (e.g., ¹H or¹³C), infrared spectroscopy, spectrophotometry (e.g., UV-visible), ormass spectrometry, or by chromatography such as high performance liquidchromatography (HPLC) or thin layer chromatography.

The expressions, “ambient temperature,” “room temperature,” and “r.t.”,as used herein, are understood in the art, and refer generally to atemperature, e.g. a reaction temperature, that is about the temperatureof the room in which the reaction is carried out, for example, atemperature from about 20° C. to about 30° C.

Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) ProbeDisplacement Assay

The PARP probes can be used in time-resolved fluorescence resonanceenergy transfer (TR-FRET) probe displacement assays. TR-FRET is thecombination of time-resolved fluorometry (TRF) with Forster resonanceenergy transfer (FRET). It can offer a powerful tool for studying theinteractions between biomolecules.

FRET occurs when a donor fluorophore in its excited state transfersenergy by a non-radiative dipole-dipole coupling mechanism to anacceptor fluorophore in close proximity (e.g., <10 nm). As a result, theacceptor emission is predominantly observed because of theintermolecular FRET from the donor to the acceptor. FRET can bequantified in cuvette-based experiments or in microscopy images on apixel-by-pixel basis. This quantification can be based directly ondetecting two emission channels under two different excitationconditions (primarily donor and primarily acceptor). However, forrobustness reasons, FRET quantification is most often based on measuringchanges in fluorescence intensity.

The biological fluids or serum commonly used in these researchapplications contain many compounds and proteins which are naturallyfluorescent. Therefore, the use of conventional, steady-statefluorescence measurement presents serious limitations in assaysensitivity.

To reduce assay interference and increase data quality, a time-resolvedFRET (TR-FRET) assay can be used to identify PARP inhibitors. TR-FRETgenerally employs a long-lifetime donor fluorophore (e.g., terbiumchelate, samarium, europium, terbium, and dysprosium) and a suitableacceptor fluorophore (fluorescein or allophycocyanin).

As shown in FIG. 1, TR-FRET can be used to identify an agent that caninhibit the binding between the PARP probe and the PARP polypeptide. Themethods involve providing a polypeptide comprising a PARP catalyticdomain. The polypeptide is labeled with a donor fluorophore. In themeantime, the PARP probe is labeled with an acceptor fluorophore. Whenthe donor fluorophore is exposed to excitation light, the energy istransferred from the donor fluorophore to the acceptor fluorophore. Thesignal produced by the acceptor fluorophore is measured. In the presenceof an agent (e.g., a PARP inhibitor) that can inhibit the bindingbetween the PARP probe and the PARP polypeptide, the PARP probe is notin close proximity with the PARP polypeptide, thus the energy cannot beeffectively transferred from the donor fluorophore to the acceptorfluorophore. Therefore, the signal produced by the acceptor fluorophorewill decrease.

In certain embodiments, an agent (e.g., a test compound) is identifiedas a PARP inhibitor if it results in a decrease in the signal producedby the acceptor fluorophore by at least 10%, at least 15%, at least 20%,at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, or at least 95% as compared tothe signal of the acceptor fluorophore in the absence of the agent orany agent that can inhibit or interfere with the binding between thePARP probe and the PARP polypeptide.

In some embodiments, the signal produced by the acceptor fluorophore canbe compared to a reference level. The reference level can be the signalproduced by the acceptor fluorophore in the absence of any agent thatcan inhibit or interfere with the binding between the PARP probe and thepolypeptide.

In certain embodiments, an agent (e.g., a test compound) is identifiedas a PARP inhibitor if it results in a decrease in the ratio of thesignal produced by the acceptor to the signal produced by the donor byat least 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, or at least 95% as compared to the ratio in the absence ofthe agent or any agent that can inhibit or interfere with the bindingbetween the PARP probe and the PARP polypeptide.

In some embodiments, the ratio of the signal produced by the acceptor tothe signal produced by the donor can be compared to a reference level.The reference level can be the ratio of the acceptor signal to the donorsignal in the absence of any agent that can inhibit or interfere withthe binding between the PARP probe and the polypeptide.

In some embodiments, the polypeptide can be labeled with a donorfluorophore, and the PARP probe can be labeled with an acceptorfluorophore.

A variety of fluorophore combinations can be used in TR-FRET. In someembodiments, lanthanide ion complexes (Ln(III) chelates or cryptates)are used. In some embodiments, the donor fluorophore is Europium³⁺, andthe acceptor fluorophore is allophycocyanin. In some embodiments, thedonor fluorophore is Terbium3+, and the acceptor fluorophore isphycoerythrin.

TR-FRET measurements can be also carried out using any suitabletechnique. For example, a microscope image of donor emission can betaken with the acceptor being present. The acceptor is then bleached,such that it is incapable of accepting energy transfer and another donoremission image is acquired. A pixel based quantification using thesecond equation in the theory section above is then possible. Analternative way of temporarily deactivating the acceptor is based on itsfluorescence saturation.

In some embodiments, the ratio between the signal produced by theacceptor and the signal produced by the donor is calculated. The %inhibition can be calculated as described below:

${\%\mspace{14mu}{inhibition}} = {100 \times \frac{{TRF}_{cmpd} - {TRF}_{\min}}{{TRF}_{\max} - {TRF}_{\min}}}$

wherein TRF_(cmpd) is the TR-FRET ratio from the compound treatedsolution, TRF_(min) is the TR-FRET ratio from a positive control andTRF_(max) is the TR-FRET ratio from the negative control (e.g.,DMSO-treated).

The % inhibition values can be plotted as a function of compoundconcentration and the following 4-parameter fit can be applied to derivethe IC₅₀ values:

$Y = {{Bottom} + \frac{\left( {{Top} - {Bottom}} \right)}{\left( {1 + \left( \frac{X}{{IC}_{50}} \right)^{{Hill}\mspace{14mu}{Coefficient}}} \right.}}$

wherein top and bottom can be normally allowed to float, but may befixed at 100 or 0 respectively in a 3-parameter fit. The HillCoefficient can be normally allowed to float but may also be fixed at 1in a 3-parameter fit. Y is the % inhibition and X is the compoundconcentration. IC₅₀ is the concentration of an inhibitor where theresponse (or binding) is reduced by half. It is a measure of the potencyof a substance in inhibiting a specific biological or biochemicalfunction. Based on the modeling, IC₅₀ can be estimated.

FRET and TR-FRET methods, protocols, techniques, and assays aredescribed generally and specifically in a number of patents and patentapplications, including, e.g., U.S. Pat. Nos. 6,908,769; 6,824,990;6,762,280; 6,689,574; 6,661,909; 6,642,001; 6,639,078; 6,472,156;6,456,734; 6,376,257; 6,348,322; 6,323,039; 6,291,201; 6,280,981;5,914,245; 5,661,035; and US 20080113444; US 2009021510; Du et al. “Atime-resolved fluorescence resonance energy transfer assay forhigh-throughput screening of 14-3-3 protein-protein interactioninhibitors.” Assay and drug development technologies 11.6 (2013):367-381; each of which is incorporated herein by reference in itsentirety.

Fluorescence Polarization Probe Displacement Assay

The PARP probes can also be used in a fluorescence polarization probedisplacement assay. Fluorescence polarization (FP) is a homogeneousmethod that allows rapid and quantitative analysis of diverse molecularinteractions and enzyme activities. This technique has been widely usedin clinical and biomedical settings, and high-throughput screening(HTS).

In fluorescence polarization assays, a fluorophore is excited withpolarized excitation light; the polarized fluorescence is then measuredthrough an emission polarizer either parallel or perpendicular to theexciting light's plane of polarization. If a fluorescent molecule isstationary and exposed to plane-polarized light, it will become excitedand consequently emit radiation back to the polarized-plane. However, ifthe excited fluorescent molecule is in motion (rotational ortranslational) during the fluorescent lifetime, it will emit light in adifferent direction than the excitation plane. The rate at which amolecule rotates is indicative of its size. When a fluorescent-labelledmolecule binds to another molecule, the rotational motion will change,resulting in an altered intensity of plane-polarized light, whichresults in altered fluorescence polarization.

As shown in FIG. 2, when the PARP probe binds to the PARP polypeptide,the complex has a relatively larger molecular size. When an agent (e.g.,PARP inhibitor) inhibits the binding between the PARP probe and the PARPpolypeptide, the PARP probe with the fluorophore will not bind to thePARP polypeptide, thus the probe can rotate more quickly, resulting in adecrease of fluorescence polarization. This change can be detected,thereby determining whether an agent is a PARP inhibitor. In certainembodiments, an agent (e.g., a test compound) is identified as a PARPinhibitor if the fluorescence polarization decreases by at least 10%, atleast 15%, at least 20%, at least 25%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, or atleast 95% as compared to the fluorescence polarization in the absence ofthe agent or any agent that can inhibit or interfere with the bindingbetween the PARP probe and the PARP polypeptide.

A detailed description of fluorescence polarization and the method ofimplementing it is described, e.g., in Lea, Wendy A., and AntonSimeonov. “Fluorescence polarization assays in small moleculescreening.” Expert opinion on drug discovery 6.1 (2011): 17-32; U.S.Pat. No. 6,432,632; US20030082665; each of which is incorporated hereinby reference in its entirety.

Bioluminescence Resonance Energy Transfer (BRET) Probe DisplacementAssay

The PARP probes can also be used in bioluminescence resonance energytransfer (BRET) probe displacement assays. A limitation of FRET is therequirement for external illumination to initiate the fluorescencetransfer, which can lead to background noise. Bioluminescence resonanceenergy transfer involves a bioluminescent luciferase (e.g., theluciferase from Renilla reniformis, or Oplophorus gracilirostris) toproduce an initial photon emission.

As compared to FRET, in BRET, the donor fluorophore is replaced by aluciferase. As shown in FIG. 3, in the presence of a substrate,bioluminescence from the luciferase excites the acceptor fluorophorethrough Forster resonance energy transfer mechanisms. Thus, if theacceptor fluorophore is in close proximity with the luciferase, theacceptor fluorophore accepts the energy from the luciferase and emits alight with different length.

When an agent (e.g., PARP inhibitor) inhibits the binding between PARPprobe and the PARP polypeptide, the PARP probe with the acceptorfluorophore will not be in close proximity with the PARP polypeptide,thus the light from the luciferase enzyme reaction cannot be transferredto the acceptor fluorophore, resulting a decrease of fluorescenceemitted from the acceptor fluorophore. This change can be detected,thereby determining whether an agent is a PARP inhibitor. In certainembodiments, an agent (e.g., a test compound) is identified as a PARPinhibitor if it results in a decrease in the fluorescence emitted fromthe acceptor fluorophore by at least 10%, at least 15%, at least 20%, atleast 25%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, or at least 95% as compared tothe fluorescence emitted from the acceptor fluorophore in the absence ofthe agent or any agent that can inhibit or interfere with the bindingbetween the PARP probe and the PARP polypeptide.

In some embodiments, the luciferase is NanoLuc, and the acceptorfluorophore is NanoBRET® 590SE.

BRET ratio can be measured b the formula as shown below:

${{BRET}\mspace{14mu}{ratio}} = \frac{{Emission}\mspace{14mu}{of}\mspace{14mu}{the}\mspace{14mu}{acceptor}\mspace{14mu}{fluorophore}}{Luminescence}$

Control wells containing a negative control or a positive control areused to calculate the % inhibition as described below:

${\%\mspace{14mu}{inhibition}} = {100 \times \frac{{{BRET}\mspace{14mu}{ratio}_{cmpd}} - {{BRET}\mspace{14mu}{ratio}_{\min}}}{{{BRET}\mspace{14mu}{ratio}_{\max}} - {{BRET}\mspace{14mu}{ratio}_{\min}}}}$

wherein BRET ratio_(cmpd) is the BRET ratio from the compound treatedsolution, BRET ratio_(min) is the BRET ratio from the positive controland BRET ratio_(max) is the BRET ratio from the negative control.

The % inhibition values are plotted as a function of compoundconcentration and the following 4-parameter fit is applied to derive theIC₅₀ values:

$Y = {{Bottom} + \frac{\left( {{Top} - {Bottom}} \right)}{\left( {1 + \left( \frac{X}{{IC}_{50}} \right)^{{Hill}\mspace{14mu}{Coefficient}}} \right.}}$

wherein top and bottom can be normally allowed to float, but may befixed at 100 or 0 respectively in a 3-parameter fit. The HillCoefficient can be normally allowed to float but may also be fixed at 1in a 3-parameter fit. Y is the % inhibition and X is the compoundconcentration. The IC₅₀ value can be derived from the modeling.

High Throughput Screening and Compound Library

The assays as described herein can be used in high throughput screeningfor PARP modulators (e.g., monoPARP inhibitors). Such assays can be usedto screen small molecule libraries available from various commercialsources. Screening of such libraries, including combinatoriallygenerated libraries (e.g., peptide libraries), is a rapid and efficientway to screen a large number of related (and unrelated) compounds foractivity.

This disclosure provides methods for screening test compounds, e.g.,polypeptides (including, e.g., antibodies and antigen-binding fragmentsthereof), polynucleotides, inorganic or organic large or small moleculetest compounds, to identify agents useful for modulating PARP enzymaticactivity, and for the treatment of disorders associated with PARPoverexpression or overactivity (e.g., cancer or inflammation).

As used herein, “small molecules” refers to small organic or inorganicmolecules of molecular weight below about 3,000 Daltons. In general,small molecules useful for the methods as described herein can have amolecular weight of less than 3,000 Daltons (Da). The small moleculescan be, e.g., from at least about 100 Da to about 3,000 Da (e.g.,between about 100 to about 3,000 Da, about 100 to about 2500 Da, about100 to about 2,000 Da, about 100 to about 1,750 Da, about 100 to about1,500 Da, about 100 to about 1,250 Da, about 100 to about 1,000 Da,about 100 to about 750 Da, about 100 to about 500 Da, about 200 to about1500, about 500 to about 1000, about 300 to about 1000 Da, or about 100to about 250 Da).

The test compounds can be, e.g., natural products or members of acombinatorial chemistry library. A set of diverse molecules should beused to cover a variety of functions such as charge, aromaticity,hydrogen bonding, flexibility, size, length of side chain,hydrophobicity, and rigidity. Combinatorial techniques suitable forsynthesizing small molecules are known in the art, e.g., as exemplifiedby Obrecht and Villalgordo, Solid-Supported Combinatorial and ParallelSynthesis of Small-Molecular-Weight Compound Libraries,Pergamon-Elsevier Science Limited (1998), and include those such as the“split and pool” or “parallel” synthesis techniques, solid-phase andsolution-phase techniques, and encoding techniques (see, for example,Czarnik, Curr. Opin. Chem. Bio. 1:60-6 (1997)). In addition, a number ofsmall molecule libraries are commercially available.

Libraries screened using the methods as described herein can comprise avariety of types of test compounds. A given library can comprise a setof structurally related or unrelated test compounds. In someembodiments, the test compounds are peptide or peptidomimetic molecules.In some embodiments, the test compounds are nucleic acids. In someembodiments, the test compounds are antibodies or antigen-bindingfragments thereof.

In some embodiments, the test compounds and libraries thereof can beobtained by systematically altering the structure of a first testcompound, e.g., a first test compound that is structurally similar to aknown natural binding partner of the target polypeptide, or a firstsmall molecule identified as capable of binding the target polypeptide,e.g., using methods known in the art or the methods described herein,and correlating that structure to a resulting biological activity, e.g.,a structure-activity relationship study. As one of skill in the art willappreciate, there are a variety of standard methods for creating such astructure-activity relationship. Thus, in some instances, the work maybe largely empirical, and in others, the three-dimensional structure ofan endogenous polypeptide or portion thereof can be used as a startingpoint for the rational design of a small molecule compound or compounds.For example, in one embodiment, a general library of small molecules isscreened, e.g., using the methods described herein.

In some embodiments, a test compound is applied to a test sample, e.g.,a cell or living tissue, e.g., tumor tissue, and one or more effects ofthe test compound is evaluated. For example, the ability of the testcompound to inhibit cell growth or tumor growth is evaluated.

In some embodiments, the test sample is, or is derived from (e.g., asample taken from) an in vivo model. For example, an animal model, e.g.,a rodent such as a rat, can be used.

Thus, test compounds identified as “hits” (e.g., test compounds that caninhibit PARP) in a first screen can be selected and systematicallyaltered, e.g., using rational design, to optimize binding affinity,avidity, specificity, or other parameter. Such optimization can also bescreened for using the methods described herein. Thus, in oneembodiment, the disclosure includes screening a first library ofcompounds using a method known in the art and/or described herein,identifying one or more hits in that library, subjecting those hits tosystematic structural alteration to create a second library of compoundsstructurally related to the hit, and screening the second library usingthe methods described herein.

Test compounds identified as hits can be considered candidatetherapeutic compounds, useful in treating disorders associated with PARPoverexpression or overactivity (e.g., cancer). A variety of techniquesuseful for determining the structures of “hits” can be used in themethods described herein, e.g., NMR, mass spectrometry, gaschromatography equipped with electron capture detectors, fluorescenceand absorption spectroscopy. Thus, the disclosure also includescompounds identified as “hits” by the methods described herein, andmethods for their administration and use in the treatment, prevention,or delay of development or progression of a disorder described herein.

Test compounds identified as candidate therapeutic compounds can befurther screened by administration to an animal model of a disorderassociated with PARP overexpression or overactivity (e.g., cancer). Theanimal can be monitored for a change in the disorder, e.g., for animprovement in a parameter of the disorder, e.g., a parameter related toclinical outcome. In some embodiments, the parameter is tumor growth,and an improvement would inhibit tumor growth.

In some embodiments, the PARP modulators obtained from the screening areinhibitors of PARP1, PARP2, PARP3, PARP4, PARP5a, PARP5b, PARP6, TIPARP,PARP8, PARP9, PARP10, PARP11, PARP12, PARP13, PARP14, PARP15, or PARP16.In some embodiments, the inhibitors have an IC₅₀ for a PARP (e.g.,PARP1, PARP2, PARP3, PARP4, PARP5a, PARP5b, PARP6, TIPARP, PARP8, PARP9,PARP10, PARP11, PARP12, PARP13, PARP14, PARP15, or PARP16) of less than1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110,120, 130, 140, 150, 160, 170, 180, 190, or 200 nM.

In some embodiments, the binding affinity of the PARP inhibitors (K_(d))(between the compound and PARP) is less than 1×10⁻⁶ M, less than 1×10⁻⁷M, less than 1×10⁻⁸ M, less than 1×10⁻⁹ M, or less than 1×10⁻¹⁰ M. Insome embodiments, the K_(d) is less than 50 nM, 30 nM, 20 nM, 15 nM, 10nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM. In someembodiments, K_(d) is greater than 1×10⁻⁷ M, greater than 1×10⁻⁸ M,greater than 1×10⁻⁹ M, greater than 1×10⁻¹⁰ M, greater than 1×10⁻¹¹ M,or greater than 1×10⁻¹² M.

Methods of Treatment

The methods described herein include methods for the treatment ofdiseases or disorders associated with PARP overexpression oroveractivity comprising administering to a patient in need thereof atherapeutically effective amount of a compound identified according toone or more of the assays described herein. In some embodiments, thedisease or disorder is cancer. Generally, the methods includeadministering a therapeutically effective amount of PARP modulators(e.g., inhibitors) identified by the methods as described herein, to asubject who is in need of, or who has been determined to be in need of,such treatment. As used in this context, to “treat” means to ameliorateat least one symptom of the disorder associated with PARP overexpressionor overactivity. Often, the treatment results in a decreased activity ofPARP.

In some embodiments, the disease or disorder is cancer. In someembodiments, administration of a therapeutically effective amount of acompound described herein can result in a decrease of tumor size ortumor volume, a decrease of tumor growth, a reduction of the increaserate of tumor volume in a subject (e.g., as compared to the rate ofincrease in tumor volume in the same subject prior to treatment or inanother subject without such treatment), a decrease in the risk ofdeveloping a metastasis or the risk of developing one or more additionalmetastasis, an increase of survival rate, and an increase of lifeexpectancy, etc.

As used herein, the term “cancer” refers to cells having the capacityfor autonomous growth, i.e., an abnormal state or conditioncharacterized by rapidly proliferating cell growth. The term is meant toinclude all types of cancerous growths or oncogenic processes,metastatic tissues or malignantly transformed cells, tissues, or organs,irrespective of histopathologic type or stage of invasiveness. The term“tumor” as used herein refers to cancerous cells, e.g., a mass ofcancerous cells.

As used herein, the terms “subject” and “patient” are usedinterchangeably throughout the specification and describe an animal,human or non-human, to whom treatment according to the methods of thepresent invention is provided. Veterinary and non-veterinaryapplications are contemplated by the present invention. Human patientscan be adult humans or juvenile humans (e.g., humans below the age of 18years old). In addition to humans, patients include but are not limitedto mice, rats, hamsters, guinea-pigs, rabbits, ferrets, cats, dogs, andprimates. Included are, for example, non-human primates (e.g., monkey,chimpanzee, gorilla, and the like), rodents (e.g., rats, mice, gerbils,hamsters, ferrets, rabbits), lagomorphs, swine (e.g., pig, miniaturepig), equine, canine, feline, bovine, and other domestic, farm, and zooanimals.

Kits and Compositions

The present disclosure also provides kits for PARP inhibitor screening.The kit can include a PARP probe comprising a fluorophore or an affinitytag. The kit can also include a polypeptide comprising a PARP catalyticdomain and optionally a fusion/affinity tag (e.g., hexahistidine). Insome embodiments, the polypeptide is labeled with a fluorophore.

The present disclosure also provides compositions for the PARPinhibitors. In some embodiments, the compositions are formulated with apharmaceutically acceptable carrier. The pharmaceutical compositions andformulations can be administered parenterally, topically, orally or bylocal administration, such as by aerosol or transdermally. Thepharmaceutical compositions can be formulated in any way and can beadministered in a variety of unit dosage forms depending upon thecondition or disease and the degree of illness, the general medicalcondition of each patient, the resulting preferred method ofadministration and the like. Details on techniques for formulation andadministration of pharmaceuticals are well described in the scientificand patent literature, see, e.g., Remington: The Science and Practice ofPharmacy, 21st ed., 2005.

EXAMPLES

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

Materials and Methods

The following equipment and methods were used in the following examples.

¹H NMR Spectra were recorded at 300 or 400 MHz using a Bruker AVANCE 400MHz spectrometer. NMR interpretation was performed using MestReC orMestReNova software to assign chemical shift and multiplicity. In caseswhere two adjacent peaks of equal or unequal height were observed, thesetwo peaks may be labeled as either a multiplet or as a doublet. In thecase of a doublet, a coupling constant using this software may beassigned. In any given example, one or more protons may not be observeddue to obscurity by water and/or solvent peaks.

Liquid chromatography-mass spectrometry (LCMS) equipment and conditionswere as follows:

-   -   1. Liquid chromatography (LC): Agilent Technologies 1290 series,        Binary Pump, Diode Array Detector. Agilent Poroshell 120 EC-C18,        2.7 μm, 4.6×50 mm column. Mobile phase: A: 0.05% Formic acid in        water (v/v), B: 0.05% Formic acid in ACN (v/v). Flow Rate: 1        mL/min at 25° C. Detector: 214 nm, 254 nm. Gradient stop time,        10 min. Timetable is shown below:

TABLE 2 T (min) A (%) B (%) 0.0 90 10 0.5 90 10 8.0 10 90 10.0 0 100

-   -   2. Mass spectrometry (MS): G6120A, Quadrupole LC/MS, Ion Source:        ES-API, TIC: 70˜1000 m/z, Fragmentor: 60, Drying gas flow: 10        L/min, Nebulizer pressure: 35 psi, Drying gas temperature: 350°        C., Vcap: 3000V.    -   3. Sample preparation: samples were dissolved in ACN or methanol        at 1˜10 mg/mL, then filtered through a 0.22 μm filter membrane.        Injection volume: 1˜10.

The following abbreviations are used in the disclosure: ACN(acetonitrile); Boc (tert-butoxycarbonyl); Boc2O (di-tert-butyldicarbonate); CuI (copper iodide); CDCl₃ (deuterated chloroform); CD₃OD(deuterated methanol); DCM (dichloromethane); DIPEA(N,N-diisopropylethylamine); DMF (N,N-dimethylformamide); DMSO(dimethylsulfoxide); DMSO-d₆ (deuterated dimethylsulfoxide); eq(equivalent); EtOAc (ethyl acetate); g (gram); h (hour); HATU(1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxide hexafluorophosphate); ¹H NMR (proton nuclear magneticresonance); HCl (hydrochloric acid); Hz (hertz); L (litre); LiCl(lithium chloride); LCMS (liquid chromatography-mass spectrometry); M(molar); MeOH (methanol); mg (milligrams); MHz (megahertz); min(minutes); mL (millilitres), mmol (millimoles); NMP(N-methyl-2-pyrrolidone); prep-HPLC (preparative high-performance liquidchromatography); Pd(OAc)2 (palladium (II) acetate); ppm (parts permillion); Pd(allyl)Cl₂ (Bis(η3-allyl)di(μ-chloro)dipalladium(II));Rockphos(2-Di(tert-butyl)phosphino-2,4,6-triisopropyl-3-methoxy-6-methylbiphenyl);RT (room temperature); SEM (2-(trimethylsilyl)ethoxymethyl); SEMCl(2-(trimethylsilyl)ethoxymethyl chloride); TEA (triethyl amine);tBuBrettphos(2-(Di-tert-butylphosphino)-2,4,6-triisopropyl-3,6-dimethoxy-1,1-biphenyl);THF (tetrahydrofuran); TLC (thin layer chromatography); v/v(volume/volume).

Example 1: Synthesis of Intermediates Int-A1:5-Chloro-4-(trifluoromethyl)-2-[[2-(trimethylsilyl)ethoxy]methyl]-2,3-dihydropyridazin-3-one

Step 1:4,5-Dibromo-2-[[2-(trimethylsilyl)ethoxy]methyl]-2,3-dihydropyridazin-3-one

To a solution of 4,5-dibromo-2,3-dihydropyridazin-3-one (3500 g, 13.78mol, 1.00 equiv) in DMF (30 L) was added sodium hydride (400 g, 16.56mol, 1.20 equiv) in batches at 0° C. under nitrogen. The resultingsolution was stirred for 1 hour at room temperature, then[2-(chloromethoxy)ethyl]trimethylsilane (2500 g, 15.2 mol, 1.10 equiv)was added dropwise at 0° C. and stirred for 2 hours at room temperature.The reaction was then quenched by the addition of 30 L of water. Theresulting solution was extracted with 3×50 L of ethyl acetate and theorganic layers combined. The organic layers were washed with 3×30 L ofbrine, dried over anhydrous sodium sulfate and concentrated underreduced pressure to afford 4.2 kg of title compound. LCMS: [M+H]⁺384.70.

Step 2:4-Bromo-5-chloro-2-[[2-(trimethylsilyl)ethoxy]methyl]-2,3-dihydropyridazin-3-one

To a solution of4,5-dibromo-2-[[2-(trimethylsilyl)ethoxy]methyl]-2,3-dihydropyridazin-3-one(2200 g, 5.73 mol, 1.00 equiv) in NMP (6 L) was added chlorolithium (231g, 5.73 mol, 1.00 equiv) and stirred for 4 hours at 95° C. The reactionwas then diluted by the addition of 10 L of water, extracted with 3×20 Lof ethyl acetate and the organic layers combined. The organic layerswere washed with 3×20 L of brine, dried over anhydrous sodium sulfateand concentrated under reduced pressure. The residue was purified bycolumn chromatography (EtOAc:petroleum ether, 1:50, v/v) to afford 4.2kg of4,5-dibromo-2-[[2-(trimethylsilyl)ethoxy]methyl]-2,3-dihydropyridazin-3-one.This was repeated 2 times resulting in 2.2 kg of4-bromo-5-chloro-2-[[2-(trimethylsilyl)ethoxy]methyl]-2,3-dihydropyridazin-3-one.LCMS: [M+H]⁺ 340.90.

Step 3:5-Chloro-4-(trifluoromethyl)-2-[[2-(trimethylsilyl)ethoxy]methyl]-2,3-dihydropyridazin-3-one

To a solution of4-bromo-5-chloro-2-[[2-(trimethylsilyl)ethoxy]methyl]-2,3-dihydropyridazin-3-one(1100 g, 3.23 mol, 1.00 equiv) in NMP (6 L) at room temperature wasadded CuI (56 g, 0.64 mol, 0.20 equiv) followed by dropwise addition ofmethyl 2,2-difluoro-2-(fluorosulfonyl)acetate (1865 g, 9.7 mol, 3.00equiv). The resulting solution was stirred for 2 hours at 80° C. Thereaction was then quenched by the addition of 10 L of water andextracted with 3×10 L of ethyl acetate. The organic layers were combinedand washed with 3×10 L of brine, dried over anhydrous sodium sulfate,and concentrated under reduced pressure. The residue was purified bycolumn chromatography (ethyl acetate/petroleum ether, 1/100, v/v) toafford 1030 g (76%) of the title compound. LCMS: [M+H]⁺ 329.00.

¹H NMR (300 MHz, CDCl₃) δ 7.82 (s, 1H), 5.50 (d, J=27.3 Hz, 2H), 3.74(dt, J=12.9, 8.2 Hz, 2H), 0.97 (td, J=8.3, 5.0 Hz, 2H), 0.01 (d, J=2.1Hz, 9H).

Int-A2:2-[4-([2-[6-Oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-4-yl]-2,3-dihydro-1H-isoindol-5-yl]oxy)piperidin-1-yl]aceticacid

Step 1:5-(5-Hydroxy-2,3-dihydro-1H-isoindol-2-yl)-4-(trifluoromethyl)-2-[[2-(trimethylsilyl)ethoxy]methyl]-2,3-dihydropyridazin-3-one

A solution of5-chloro-4-(trifluoromethyl)-2-[[2-(trimethylsilyl)ethoxy]methyl]-2,3-dihydropyridazin-3-one(2.8 g, 8.52 mmol, 1.00 equiv), 2,3-dihydro-1H-isoindol-5-olhydrobromide (4.27 g, 19.76 mmol, 1.00 equiv), and TEA (10 mL) inethanol (40 mL) was stirred for 1 h at 60° C. The resulting solution wasextracted with 2×100 mL of ethyl acetate and the organic layers combinedand concentrated under reduced pressure to afford 4.5 g of the titlecompound as a yellow oil. LCMS: [M+H]⁺ 428.23.

Step 2: tert-Butyl4-([2-[6-oxo-5-(trifluoromethyl)-1-[[2-(trimethylsilyl)ethoxy]methyl]-1,6-dihydropyridazin-4-yl]-2,3-dihydro-1H-isoindol-5-yl]oxy)piperidine-1-carboxylate

A solution of5-(5-hydroxy-2,3-dihydro-1H-isoindol-2-yl)-4-(trifluoromethyl)-2-[[2-(trimethylsilyl)ethoxy]methyl]-2,3-dihydropyridazin-3-one(4.5 g, 10.53 mmol, 1.00 equiv), tert-butyl4-iodopiperidine-1-carboxylate (20 g, 64.28 mmol, 8.00 equiv), potassiumcarbonate (15 g, 108.53 mmol, 10.00 equiv), and DMF (50 mL) was stirredfor 2 days at 80° C. The resulting solution was extracted with 2×200 mLof ethyl acetate and the organic layers combined and concentrated underreduced pressure. The residue was applied onto a silica gel columneluting with ethyl acetate/petroleum ether to afford the title compound(2 g, 31%) as a yellow oil. LCMS: [M+H]⁺ 611.15.

Step 3:5-[5-(Piperidin-4-yloxy)-2,3-dihydro-1H-isoindol-2-yl]-4-(trifluoromethyl)-2-[[2-(trimethylsilyl)ethoxy]methyl]-2,3-dihydropyridazin-3-one

A solution of tert-butyl4-([2-[6-oxo-5-(trifluoromethyl)-1-[[2-(trimethylsilyl)ethoxy]methyl]-1,6-dihydropyridazin-4-yl]-2,3-dihydro-1H-isoindol-5-yl]oxy)piperidine-1-carboxylate(2 g, 3.27 mmol, 1.00 equiv), dioxane/HCl (5 mL), and dioxane (45 mL)was stirred for 6 h at 25° C. The resulting mixture was concentratedunder reduced pressure. The residue was applied onto a silica gel columnand eluted with ethyl acetate/petroleum ether to afford 1 g of titlecompound as a yellow oil. LCMS: [M+H]⁺ 511.28.

Step 4: tert-Butyl2-[4-([2-[6-oxo-5-(trifluoromethyl)-1-[[2-(trimethylsilyl)ethoxy]methyl]-1,6-dihydropyridazin-4-yl]-2,3-dihydro-1H-isoindol-5-yl]oxy)piperidin-1-yl]acetate

A solution of5-[5-(piperidin-4-yloxy)-2,3-dihydro-1H-isoindol-2-yl]-4-(trifluoromethyl)-2-[[2-(trimethylsilyl)ethoxy]methyl]-2,3-dihydropyridazin-3-one(1 g, 1.96 mmol, 1.00 equiv), tert-butyl 2-chloroacetate (450 mg, 2.99mmol, 3.00 equiv), DIPEA (5 mL), and dichloromethane (10 mL) was stirredovernight at 25° C. The residue was purified by C18 reverse phasechromatography eluting with H₂O/CH₃CN to afford the title compound (540mg, 44%) as a yellow oil. LCMS: [M+H]⁺ 625.20.

Step 5:2-[4-([2-[6-Oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-4-yl]-2,3-dihydro-1H-isoindol-5-yl]oxy)piperidin-1-yl]aceticacid

A solution of tert-butyl2-[4-([2-[6-oxo-5-(trifluoromethyl)-1-[[2-(trimethylsilyl)ethoxy]methyl]-1,6-dihydropyridazin-4-yl]-2,3-dihydro-1H-isoindol-5-yl]oxy)piperidin-1-yl]acetate(540 mg, 0.86 mmol, 1.00 equiv) and dioxane/HCl (8 mL) was stirredovernight at 25° C. The resulting mixture was concentrated under reducedpressure. The residue was purified by C18 reverse phase chromatographyeluting with H₂O/CH₃CN to afford 200 mg (53%) of title compound as awhite solid. LCMS: [M+H]⁺ 439.31.

Example 2: Synthesis of Probe A

Step 1: tert-ButylN-(6-[2-[4-([2-[6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-4-yl]-2,3-dihydro-1H-isoindol-5-yl]oxy)piperidin-1-yl]acetamido]hexyl)carbamate

A solution of2-[4-([2-[6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-4-yl]-2,3-dihydro-1H-isoindol-5-yl]oxy)piperidin-1-yl]aceticacid (Int-A2) (44 mg, 0.10 mmol, 1.00 equiv), DIPEA (52 mg, 0.40 mmol,4.00 equiv), HATU (46 mg, 0.12 mmol, 1.20 equiv), and tert-butylN-(6-aminohexyl)carbamate (24 mg, 0.11 mmol, 1.10 equiv) in DMF (1 mL)was stirred overnight at 25° C. The crude product was purified by C18reverse phase chromatography eluting with H₂O/CH₃CN to afford 38 mg(59%) of title compound as an off-white solid. LCMS: [M+H]⁺ 637.31.

Step 2:N-(6-Aminohexyl)-2-[4-([2-[6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-4-yl]-2,3-dihydro-1H-isoindol-5-yl]oxy)piperidin-1-yl]acetamidehydrochloride

A solution of tert-butylN-(6-[2-[4-([2-[6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-4-yl]-2,3-dihydro-1H-isoindol-5-yl]oxy)piperidin-1-yl]acetamido]hexyl)carbamate(38 mg, 0.06 mmol, 1.00 equiv) in hydrogen chloride/dioxane (10 mL) wasstirred for 3 hours at 25° C. The resulting mixture was concentratedunder reduced pressure to afford the title compound as a gray solid (30mg, 88%). LCMS: [M-Cl]⁺: 537.27.

Step 3:2,2-Difluoro-4-[2-[(6-[2-[4-([2-[6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-4-yl]-2,3-dihydro-1H-isoindol-5-yl]oxy)piperidin-1-yl]acetamido]hexyl)carbamoyl]ethyl]-12-(1H-pyrrol-2-yl)-1{circumflexover ( )}[5],3-diaza-2{circumflex over( )}[4]-boratricyclo[7.3.0.0{circumflex over( )}[3,7]]dodeca-1(12),4,6,8,10-pentaen-1-yliumc

A solution of NanoBRET® 590SE(N-(6-aminohexyl)-2-[4-([2-[6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-4-yl]-2,3-dihydro-1H-isoindol-5-yl]oxy)piperidin-1-yl]acetamidehydrochloride (23 mg, 0.04 mmol, 2.00 equiv), DIPEA (52 mg, 0.40 mmol,5.00 equiv),4-3-[(2,5-dioxopyrrolidin-1-yl)oxy]-3-oxopropyl-2,2-difluoro-12-(1H-pyrrol-2-yl)-1{circumflexover ( )}5,3-diaza-2{circumflex over( )}4-boratricyclo[7.3.0.0{circumflex over( )}3,7]dodeca-1(12),4,6,8,10-pentaen-1-ylium) (10 mg, 0.02 mmol, 1.00equiv) in dichloromethane (2 mL) and methanol (2 mL) was stirred for 2 hat 25° C. The resulting mixture was concentrated under reduced pressureand the crude product was purified by C18 reverse phase chromatographyeluting with H₂O/CH₃CN to afford 6.9 mg of a purple solid (35%). LCMS:[M+H]⁺ 848.38.

¹H NMR (CD₃OD, 400 MHz) δ: 7.98 (s, 1H), 7.28-7.14 (m, 5H), 7.02-6.84(m, 4H), 6.37-6.26 (m, 2H), 4.93 (d, J=12.0 Hz, 4H), 4.45-4.35 (m, 1H),3.29-3.13 (m, 6H), 3.01 (s, 2H), 2.81-2.70 (m, 2H), 2.60 (t, J=7.7 Hz,2H), 2.43 (td, J=8.7, 4.6 Hz, 2H), 2.01 (dd, J=11.8, 7.0 Hz, 2H), 1.81(ddt, J=15.8, 11.5, 5.5 Hz, 2H), 1.51 (q, J=7.3, 6.8 Hz, 4H), 1.38-1.26(m, 4H).

Example 3: Synthesis of Probe B

Step 1: tert-ButylN-(6-[2-[4-([2-[6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-4-yl]-2,3-dihydro-1H-isoindol-5-yl]oxy)piperidin-1-yl]acetamido]hexyl)carbamate

A solution of2-[4-([2-[6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-4-yl]-2,3-dihydro-1H-isoindol-5-yl]oxy)piperidin-1-yl]aceticacid (Int-A2) (250 mg, 0.57 mmol, 1.00 equiv), tert-butylN-(6-aminohexyl)carbamate (120 mg, 0.55 mmol, 1.00 equiv), HATU (220 mg,0.58 mmol, 1.10 equiv), DIPEA (2 mL), and DMF (4 mL) was stirred for 0.5h at 0° C. The residue was purified by C18 reverse phase chromatographyeluting with H₂O/CH₃CN to afford 190 mg (52%) of the title compound as awhite solid. LCMS: [M+H]⁺ 637.32.

Step 2:N-(6-Aminohexyl)-2-[4-([2-[6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-4-yl]-2,3-dihydro-1H-isoindol-5-yl]oxy)piperidin-1-yl]acetamidehydrochloride

A solution of tert-butylN-(6-[2-[4-([2-[6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-4-yl]-2,3-dihydro-1H-isoindol-5-yl]oxy)piperidin-1-yl]acetamido]hexyl)carbamate(190 mg, 0.30 mmol, 1.00 equiv) and dioxane/HCl (6 mL) was stirred for 1hour at 25° C. The resulting mixture was concentrated under reducedpressure. The residue was purified by C18 reverse phase chromatographyeluting with H₂O/CH₃CN to afford 100 mg (58%) of title compound as ayellow oil. LCMS: [M+H]⁺ 537.27.

Step 3:17-[2-Carboxylato-5-[(6-[2-[4-([2-[6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-4-yl]-2,3-dihydro-1H-isoindol-5-yl]oxy)piperidin-1-yl]acetamido]hexyl)carbamoyl]phenyl]-3-oxa-9{circumflexover ( )}[5],25-diazaheptacyclo[18.8.1.{circumflex over( )}[5,9].0{circumflex over ( )}[2,18].0{circumflex over( )}[4,16].0{circumflex over ( )}[25,29].0{circumflex over( )}[14,30]]triaconta-1(29),2(18),4,9(30),14,16,19-heptaen-9-ylium

A solution ofN-(6-aminohexyl)-2-[4-([2-[6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-4-yl]-2,3-dihydro-1H-isoindol-5-yl]oxy)piperidin-1-yl]acetamidehydrochloride (8 mg, 0.01 mmol, 1.00 equiv),17-[2-carboxylato-5-(2,3,5,6-tetrafluorophenoxycarbonyl)phenyl]-3-oxa-9{circumflexover ( )}5,25-diazaheptacyclo[18.8.1.1{circumflex over( )}5,9.0{circumflex over ( )}2,18.0{circumflex over( )}4,16.0{circumflex over ( )}25,29.0{circumflex over( )}14,30]triaconta-1(29),2(18),4,9(30),14,16,19-heptaen-9-ylium(NanoBRET® 618TFP Ester) (10 mg, 0.01 mmol, 1.00 equiv), DIPEA (0.8 mL),and DMF (6 mL) was stirred for 1 h at 25° C. The resulting mixture wasconcentrated under reduced pressure. The crude product was purified byPrep-HPLC (XBridge Prep C18 OBD column, 5 μm, 19×150 mm column, elutingwith water:acetonitrile (50:50, v:v) with 0.1% NH₄HCO₃, at a flow rateof 1.2 mL/min) to afford the title compound as a blue solid (0.8 mg,5%). LCMS: [M+H]⁺ 1081.45.

¹H NMR (CD₃OD, 400 MHz) δ: 8.09-8.02 (m, 3H), 7.68 (d, J=1.6 Hz, 1H),7.22 (d, J=8.4 Hz, 1H), 6.93-6.83 (m, 4H), 4.91 (s, 4H), 4.55-4.51 (m,1H), 3.72-3.69 (m, 3H), 3.49-3.54 (m, 3H), 3.35-3.42 (m, 2H), 3.27-3.23(m, 2H), 3.15-3.01 (m, 5H), 2.84-2.81 (m, 5H), 2.45-2.41 (m, 2H),2.08-1.92 (m, 15H), 1.84-1.80 (m, 4H), 1.66-1.37 (m, 7H), 0.92-0.85 (m,2H).

Example 4: Synthesis of Probe C

Step 1: Tert-butylN-(6-[5-[(3aS,4S,6aR)-2-oxo-hexahydro-1H-thieno[3,4-d]imidazolidin-4-yl]pentanamido]hexyl)carbamate

A solution of5-[(3aS,4S,6aR)-2-oxo-hexahydro-1H-thieno[3,4-d]imidazolidin-4-yl]pentanoicacid ((reagent was purchased from Beijing Dragon Rui Trading Company,976 mg, 3.99 mmol, 1.00 equiv), DIPEA (1.55 g, 11.99 mmol, 3.00 equiv),HATU (1.82 g, 4.79 mmol, 1.20 equiv), and tert-butylN-(6-aminohexyl)carbamate (864 mg, 3.99 mmol, 1.00 equiv) in DMF (15 mL)was stirred overnight at 25° C. The reaction was then quenched by theaddition of 50 mL of water. The solids were collected by filtration toafford 1.5 g (85%) of the title compound as a white solid. LCMS: [M+H]⁺443.26.

Step 2:5-[(3aS,4S,6aR)-2-Oxo-hexahydro-1H-thieno[3,4-d]imidazolidin-4-yl]-N-(6-aminohexyl)pentanamidehydrochloride

A solution of tert-butylN-(6-[5-[(3aS,4S,6aR)-2-oxo-hexahydro-1H-thieno[3,4-d]imidazolidin-4-yl]pentanamido]hexyl)carbamate(800 mg, 1.81 mmol, 1.00 equiv) in hydrogen chloride/dioxane (20 mL) wasstirred overnight at 25° C. The resulting mixture was concentrated underreduced pressure to afford 600 mg (88%) of the title compound as a graycrude oil. LCMS: [M+H]⁺ 343.21.

Step 3:5-[(3aS,4S,6aR)-2-Oxo-hexahydro-1H-thieno[3,4-d]imidazolidin-4-yl]-N-(6-[2-[4-([2-[6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-4-yl]-2,3-dihydro-1H-isoindol-5-yl]oxy)piperidin-1-yl]acetamido]hexyl)pentanamide

A solution of2-[4-([2-[6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-4-yl]-2,3-dihydro-1H-isoindol-5-yl]oxy)piperidin-1-yl]aceticacid (175 mg, 0.40 mmol, 1.00 equiv), DIPEA (258 mg, 2.00 mmol, 5.00equiv), HATU (228 mg, 0.60 mmol, 1.50 equiv),5-[(3aS,4S,6aR)-2-oxo-hexahydro-1H-thieno[3,4-d]imidazolidin-4-yl]-N-(6-aminohexyl)pentanamidehydrochloride (228 mg, 0.60 mmol, 1.50 equiv) in DMF (3 mL) was stirredfor 4 h at 25° C. The crude product was purified by C18 reverse phasechromatography eluting with H₂O/CH₃CN to afford the title compound as awhite solid (118.3 mg, 39%). LCMS: [M+H]⁺ 763.35.

¹H NMR (DMSO-d₆, 400 MHz) δ:12.52 (s, 1H), 7.98 (s, 1H), 7.81-7.68 (m,2H), 7.26 (d, J=8.4 Hz, 1H), 7.00 (d, J=2.2 Hz, 1H), 6.91 (dd, J=8.4,2.3 Hz, 1H), 6.45-6.39 (m, 1H), 6.36 (s, 1H), 4.91 (d, J=6.1 Hz, 4H),4.45 (m, 1H), 4.26 (m, 1H), 4.17-4.08 (m, 1H), 3.14-2.96 (m, 5H), 2.91(s, 2H), 2.82 (dd, J=12.4, 5.1 Hz, 1H), 2.73-2.63 (m, 2H), 2.58 (d,J=12.4 Hz, 1H), 2.33 (ddd, J=11.8, 9.4, 3.1 Hz, 2H), 2.11-1.90 (m, 4H),1.76-1.54 (m, 3H), 1.57-1.20 (m, 13H).

Example 5: Synthesis of Probe D

Step 1: Tert-butyl 2-(4-hydroxypiperidin-1-yl)acetate

A solution of piperidin-4-ol (10.1 g, 99.85 mmol, 1.00 equiv), DIPEA(14.2 g, 109.87 mmol, 1.10 equiv), tert-butyl 2-chloroacetate (14.5 g,96.28 mmol, 1.00 equiv) in THF (500 mL) was stirred overnight at 25° C.The solids were filtered and concentration under reduced pressureafforded the crude residue which was purified by silica gelchromatography eluting with EtOAC/petroleum ether (1/1) to afford 10.2 g(47%) of the title compound as a white solid. LCMS: [M+H]⁺ 216.15.

Step 2: Tert-butyl 2-[4-(methanesulfonyloxy)piperidin-1-yl]acetate

A solution of tert-butyl 2-(4-hydroxypiperidin-1-yl)acetate (10.2 g,47.38 mmol, 1.00 equiv), TEA (9.53 g, 94.18 mmol, 2.00 equiv), Ms₂O(9.86 g, 1.20 equiv) in DCM (200 mL) was stirred for 3 hours at 25° C.The reaction was then quenched by the addition of 300 mL of water. Theresulting solution was extracted with 200 mL of dichloromethane and theorganic layers were combined. The resulting mixture was concentratedunder reduced pressure to afford 11 g (79%) of title compound as anoff-white solid. LCMS: [M+H]⁺ 294.13.

Step 3: Tert-butyl2-[4-([2-[6-oxo-5-(trifluoromethyl)-1-[[2-(trimethylsilyl)ethoxy]methyl]-1,6-dihydropyridazin-4-yl]-2,3-dihydro-1H-isoindol-5-yl]oxy)piperidin-1-yl]acetate

A solution of5-(5-hydroxy-2,3-dihydro-1H-isoindol-2-yl)-4-(trifluoromethyl)-2-[[2-(trimethylsilyl)ethoxy]methyl]-2,3-dihydropyridazin-3-one(4.27 g, 9.99 mmol, 1.00 equiv), potassium carbonate (6.9 g, 49.92 mmol,5.00 equiv), tert-butyl 2-[4-(methanesulfonyloxy)piperidin-1-yl]acetate(5.86 g, 19.97 mmol, 2.00 equiv) in DMF (50 mL) was stirred for 2 daysat 80° C. in an oil bath. The reaction was then quenched by the additionof 100 mL of water. The resulting solution was extracted with 4×100 mLof EtOAc and the organic layers were combined. After concentration, theresidue was purified by C18 reverse phase chromatography eluting withH₂O/CH₃CN to afford 1.5 g (24%) of the title compound as a light yellowsolid. LCMS: [M+H]⁺ 625.30.

Step 4:2-[4-([2-[6-Oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-4-yl]-2,3-dihydro-1H-isoindol-5-yl]oxy)piperidin-1-yl]aceticacid

A solution of tert-butyl2-[4-([2-[6-oxo-5-(trifluoromethyl)-1-[[2-(trimethylsilyl)ethoxy]methyl]-1,6-dihydropyridazin-4-yl]-2,3-dihydro-1H-isoindol-5-yl]oxy)piperidin-1-yl]acetate(1.5 g, 2.40 mmol, 1.00 equiv) in hydrogen chloride/dioxane (40 mL) wasstirred overnight at 25° C. The resulting mixture was concentrated undervacuum. The crude product was purified by C18 reverse phasechromatography eluting with H₂O/CH₃CN to afford 600 mg (57%) of thetitle compound as a gray solid. LCMS: [M+H]⁺ 439.15.

Step 5: Tert-butylN-(6-[3′,6′-dihydroxy-3-oxo-3H-spiro[2-benzofuran-1,9′-xanthene]-6-ylformamido]hexyl)carbamate

A solution of3′,6′-dihydroxy-3-oxo-3H-spiro[2-benzofuran-1,9′-xanthene]-6-carboxylicacid (752 mg, 2.00 mmol, 1.00 equiv), DIPEA (774 mg, 5.99 mmol, 3.00equiv), HATU (912 mg, 2.40 mmol, 1.20 equiv), tert-butylN-(6-aminohexyl)carbamate (475 mg, 2.20 mmol, 1.00 equiv) in DMF (10 mL)was stirred for 3 h at 25° C. The reaction was then quenched by theaddition of 50 mL of water. The solids were filtered and the crudeproduct was purified by C18 reverse phase chromatography eluting withH₂O/CH₃CN to afford 400 mg (35%) of the title compound as a yellowsolid. LCMS: [M+H]⁺ 575.23.

Step 6:N-(6-Aminohexyl)-3′,6′-dihydroxy-3-oxo-3H-spiro[2-benzofuran-1,9′-xanthene]-6-carboxamide

A solution of tert-butylN-(6-[3′,6′-dihydroxy-3-oxo-3H-spiro[2-benzofuran-1,9′-xanthene]-6-ylformamido]hexyl)carbamate(400 mg, 0.70 mmol, 1.00 equiv) in HCl/dioxane (20 mL) was stirredovernight at 25° C. The resulting mixture was concentrated under reducedpressure and the crude product was purified by C18 reverse phasechromatography eluting with H₂O/CH₃CN to afford 200 mg of the titlecompound (61%) as a yellow solid. LCMS: [M+H]⁺ 475.18.

Step 7.N-(6-[3-Dihydroxy-3-oxo-3H-spiro[2-benzofuran-1,9′-xanthene]]-6-ylformamido]hexyl)-2-[4-([2-[6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-4-yl]-2,3-dihyro-H-isoindol-5-yl]oxy)piperidin-1-yl]acetamide

A solution of2-[4-([2-[6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-4-yl]-2,3-dihydro-1H-isoindol-5-yl]oxy)piperidin-1-yl]aceticacid (Int-A2) (150 mg, 0.34 mmol, 1.00 equiv), DIPEA (176 mg, 1.36 mmol,4.00 equiv), HATU (183 mg, 0.48 mmol, 1.40 equiv),N-(6-aminohexyl)-3-dihydroxy-3-oxo-3H-spiro[2-benzofuran-1,9′-xanthene]-6-carboxamide(228 mg, 0.48 mmol, 1.40 equiv) in DMF (5 mL) was stirred for 3 h at 25°C. After concentration, the residue was purified by C18 reverse phasechromatography eluting with H₂O/CH₃CN to afford the title compound as anorange solid (26.9 mg, 9%). LCMS: [M+H]⁺ 895.32.

¹H NMR (DMSO-d₆, 400 MHz) δ: 8.61 (t, J=5.6 Hz, 1H), 8.14-8.02 (m, 2H),7.96 (s, 1H), 7.72-7.61 (m, 2H), 7.22 (d, J=8.4 Hz, 1H), 6.96 (d, J=2.2Hz, 1H), 6.86 (dd, J=8.4, 2.2 Hz, 1H), 6.58 (d, J=8.8 Hz, 4H), 6.49 (s,2H), 4.88 (d, J=5.4 Hz, 4H), 4.34 (dp, J=8.2, 3.7 Hz, 1H), 3.17 (q,J=6.2 Hz, 2H), 3.03 (q, J=6.6 Hz, 2H), 2.86 (s, 2H), 2.70-2.58 (m, 2H),2.33-2.22 (m, 2H), 1.91 (d, J=13.5 Hz, 2H), 1.65 (dtd, J=12.6, 8.8, 3.2Hz, 2H), 1.39 (dq, J=27.4, 6.8, 6.2 Hz, 4H), 1.22 (d, J=6.8 Hz, 4H).

Example 6: Synthesis of Compound A5-[5-(Piperidin-4-yloxy)-2,3-dihydro-1H-isoindol-2-yl]-4-(trifluoromethyl)-2,3-dihydropyridazin-3-one

Step 1:5-(5-Hydroxy-2,3-dihydro-1H-isoindol-2-yl)-4-(trifluoromethyl)-2-[[2-(trimethylsilyl)ethoxy]methyl]-2,3-dihydropyridazin-3-one

A solution of Int-A1 (2.8 g, 8.52 mmol, 1.00 equiv),2,3-dihydro-1H-isoindol-5-ol hydrobromide (4.27 g, 19.76 mmol, 1.00equiv), and TEA (10 mL) in ethanol (40 mL) was stirred for 1 h at 60° C.The resulting solution was extracted with 2×100 mL of EtOAc and theorganic layers combined and concentrated under reduced pressure toafford 4.5 g of the title compound as a yellow oil. LCMS: [M+H]⁺ 428.23.

Step 2: tert-Butyl4-([2-[6-oxo-5-(trifluoromethyl)-1-[[2-(trimethylsilyl)ethoxy]methyl]-1,6-dihydropyridazin-4-yl]-2,3-dihydro-1H-isoindol-5-yl]oxy)piperidine-1-carboxylate

A solution of5-(5-hydroxy-2,3-dihydro-1H-isoindol-2-yl)-4-(trifluoromethyl)-2-[[2-(trimethylsilyl)ethoxy]methyl]-2,3-dihydropyridazin-3-one(4.5 g, 10.53 mmol, 1.00 equiv), tert-butyl4-iodopiperidine-1-carboxylate (20 g, 64.28 mmol, 8.00 equiv), potassiumcarbonate (15 g, 108.53 mmol, 10.00 equiv), and DMF (50 mL) was stirredfor 2 days at 80° C. The resulting solution was extracted with 2×200 mLof EtOAc and the organic layers combined and concentrated under reducedpressure. The residue was applied onto a silica gel column eluting withEtOAc/petroleum ether to afford the title compound (2 g, 31%) as ayellow oil. LCMS: [M+H]⁺ 611.15.

Step 3:5-[5-(Piperidin-4-yloxy)-2,3-dihydro-1H-isoindol-2-yl]-4-(trifluoromethyl)-2,3-dihydropyridazin-3-one

A solution of tert-butyl4-([2-[6-oxo-5-(trifluoromethyl)-1-[[2-(trimethylsilyl)ethoxy]methyl]-1,6-dihydropyridazin-4-yl]-2,3-dihydro-1H-isoindol-5-yl]oxy)piperidine-1-carboxylate(150 mg, 0.25 mmol, 1.00 equiv) in HCl/dioxane (5 mL) was stirredovernight at 45° C. The resulting mixture was concentrated under reducedpressure and the crude product was purified by C18 reverse phasechromatography eluting with H₂O/ACN to afford the title compound as awhite solid LCMS: [M+H]⁺ 381.28. ¹H NMR (400 MHz, Methanol-d₄) δ 8.05(s, 1H), 7.27 (d, J=8.4 Hz, 1H), 7.02-6.91 (m, 2H), 5.00 (d, J=10.6 Hz,4H), 4.61-4.48 (m, 1H), 3.21-3.10 (m, 2H), 2.89-2.78 (m, 2H), 2.11-2.08(m, 2H), 1.82-1.69 (m, 2H).

Example 7: Synthesis of Compound B6-[4-[(3-[[(1S)-2-[6-Oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-4-yl]-2,3-dihydro-1H-isoindol-1-yl]methoxy]phenyl)carbonyl]piperazin-1-yl]pyridine-3-carbonitrile

Step 1:5-[1-(Hydroxymethyl)-2,3-dihydro-1H-isoindol-2-yl]-4-(trifluoromethyl)-2-[[2-(trimethylsilyl)ethoxy]methyl]-2,3-dihydropyridazin-3-one

A solution of5-chloro-4-(trifluoromethyl)-2-[2-(trimethylsilyl)ethoxy]methyl-2,3-dihydropyridazin-3-one(4.8 g, 14.60 mmol, 1.00 equiv), 2,3-dihydro-1H-isoindol-1-ylmethanolhydrochloride (2.7 g, 14.54 mmol, 1.00 equiv) and TEA (4.4 g, 43.48mmol, 2.99 equiv) in ethanol (100 mL) was stirred for 1 h at 60° C., andthen the resulting solution was concentrated under vacuum and theresidue was applied onto a silica gel column eluting withEtOAc/petroleum ether (45:55) to afford 2.9 g (45%) of the titlecompound as a brown solid. LCMS: [M+H]⁺ 442.17.

Step 2: Methyl3-([2-[6-oxo-5-(trifluoromethyl)-1-[[2-(trimethylsilyl)ethoxy]methyl]-1,6-dihydropyridazin-4-yl]-2,3-dihydro-H-isoindol-1-yl]methoxy)benzoate

Under nitrogen, a solution of5-[1-(hydroxymethyl)-2,3-dihydro-1H-isoindol-2-yl]-4-(trifluoromethyl)-2-[[2-(trimethylsilyl)ethoxy]methyl]-2,3-dihydropyridazin-3-one(2.93 g, 6.64 mmol, 1.00 equiv), methyl 3-bromobenzoate (2.84 g, 13.21mmol, 1.99 equiv), Pd(allyl)C12 (243 mg), Rockphos (311 mg) and Cs₂CO₃(4.3 g, 13.20 mmol, 1.99 equiv) in Toluene (100 mL) was stirred for 18 hat 80° C. The resulting solution was concentrated under vacuum and thenthe residue was applied onto a silica gel column eluting withEtOAc/petroleum ether (1:3) to afford 3 g (79%) of the title compound asa brown solid. LCMS: [M+H]⁺ 576.21.

Step 3:3-([2-[6-Oxo-5-(trifluoromethyl)-1-[[2-(trimethylsilyl)ethoxy]methyl]-1,6-dihydropyridazin-4-yl]-2,3-dihydro-1H-isoindol-1-yl]methoxy)benzoicacid

A solution of methyl3-([2-[6-oxo-5-(trifluoromethyl)-1-[[2-(trimethylsilyl)ethoxy]methyl]-1,6-dihydropyridazin-4-yl]-2,3-dihydro-1H-isoindol-1-yl]methoxy)benzoate(1.15 g, 2.00 mmol, 1.00 equiv) and LiOH (240 mg, 10.02 mmol, 5.02equiv) in THF (12 mL) and water (3 mL) was stirred for 3 h at 60° C. Theresulting solution was concentrated under vacuum and the residue wasdiluted with 10 mL of H₂O, and then the pH value of the solution wasadjusted to 5 with HCl (36.5%). The solid was collected by filtration toafford 1.1 g (98%) of the title compound as a light yellow solid. LCMS:[M+H]⁺ 562.19.

Step 4:3-([2-[6-Oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-4-yl]-2,3-dihydro-1H-isoindol-1-yl]methoxy)benzoicacid

A solution of3-([2-[6-oxo-5-(trifluoromethyl)-1-[[2-(trimethylsilyl)ethoxy]methyl]-1,6-dihydropyridazin-4-yl]-2,3-dihydro-1H-isoindol-1-yl]methoxy)benzoicacid (1.1 g, 1.96 mmol, 1.00 equiv) in HCl/dioxane (20 mL, 4M) wasstirred for 3 h at RT, and then the resulting solution was concentratedunder vacuum to afford 1 g of the title compound as a crude brown solid.LCMS: [M+H]⁺ 432.11.

Step 5:6-[4-[(3-[[(1R)-2-[6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-4-yl]-2,3-dihydro-1H-isoindol-1-yl]methoxy]phenyl)carbonyl]piperazin-1-yl]pyridine-3-carbonitrileand6-[4-[(3-[[(S)-2-[6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-4-yl]-2,3-dihydro-1H-isoindol-1-yl]methoxy]phenyl)carbonyl]piperazin-1-yl]pyridine-3-carbonitrile

A solution of3-([2-[6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-4-yl]-2,3-dihydro-1H-isoindol-1-yl]methoxy)benzoicacid (500 mg, 1.16 mmol, 1.00 equiv), HATU (528 mg, 1.39 mmol, 1.20equiv), DIPEA (449 mg, 3.47 mmol, 3.00 equiv) and Int-A4 (240 mg, 1.27mmol, 1.1 equiv) in DMF (5 mL) was stirred for 2 h at RT. Afterconcentration by reduced pressure, the resulting solution was purifiedby C18 reverse phase chromatography eluting with H₂O/ACN. The residuewas further purified by Prep-HPLC and Chiral-Prep-HPLC (CHIRAL RepairedIA, 5 μm, 0.46×10 cm column, eluting with a gradient of(Hexanes:DCM=3:1)(0.1% DEA):EtOH=50:50, at a flow rate of 1 mL/min)yielding the title compound as a white solid. The absolutestereochemistry was assigned based on an X-ray crystal structure whichconfirmed (S)-absolute stereochemistry.

LCMS: [M+H]⁺ 602.05, ¹H NMR (300 MHz, Methanol-d₄) δ 8.43 (d, J=1.8 Hz,1H), 8.42 (s, 1H), 7.79 (dd, J=9.0, 2.4 Hz, 1H), 7.53-7.50 (m, 1H),7.41-7.35 (m, 4H), 7.05-6.99 (m, 2H), 6.94-6.87 (m, 2H), 6.20 (s, 1H),5.33 (d, J=14.8 Hz, 1H), 4.68 (d, J=14.7 Hz, 1H), 4.53 (dd, J=10.2, 3.3Hz, 1H), 4.29 (dd, J=10.2, 6.6 Hz, 1H), 3.91-3.44 (m, 8H). tR=5.955 min.

Example 8: Synthesis of Compound C5-[2-[(1-Acetylpiperidin-4-yl)oxy]-5H,6H,7H-pyrrolo[3,4-b]pyridin-6-yl]-4-(trifluoromethyl)-2,3-dihydropyridazin-3-one

Step 1:5-[2-Chloro-5H,6H,7H-pyrrolo[3,4-b]pyridin-6-yl]-4-(trifluoromethyl)-2-[[2-(trimethylsilyl)ethoxy]methyl]-2,3-dihydropyridazin-3-one

A solution of 2-chloro-5H,6H,7H-pyrrolo[3,4-b]pyridin-6-yl hydrochloride(5 g, 26.31 mmol, 1.00 equiv), TEA (8 g, 79.06 mmol, 3.00 equiv), andInt-A1 (14.3 g, 43.49 mmol, 1.00 equiv) in EtOH (30 mL) was stirred for2 h at 80° C. After concentration under reduced pressure, the residuewas applied onto a silica gel column with EtOAc/petroleum ether (1:4) toafford 9.3 g (79%) of title compound as a yellow oil. LCMS: [M+H]⁺447.15.

Step 2:5-[2-Hydroxy-5H,6H,7H-pyrrolo[3,4-b]pyridin-6-yl]-4-(trifluoromethyl)-2-[[2-(trimethylsilyl)ethoxy]methyl]-2,3-dihydropyridazin-3-one

A solution of5-[2-chloro-5H,6H,7H-pyrrolo[3,4-b]pyridin-6-yl]-4-(trifluoromethyl)-2-[[2-(trimethylsilyl)ethoxy]methyl]-2,3-dihydropyridazin-3-one(300 mg, 0.67 mmol, 1.00 equiv), tBuBrettphos (49 mg, 0.15 equiv), K₃PO₄(427 mg, 2.01 mmol, 3.00 equiv), and Pd(OAc)₂ (15 mg, 0.07 mmol, 0.10equiv) in dioxane (5 mL) and water (0.5 mL) was stirred for 2 h at 80°C. in an oil bath under N₂ atmosphere. After concentration, the residuewas applied onto a silica gel column with DCM/methanol (85:15) to afford200 mg (70%) of title compound as a yellow oil. LCMS: [M+H]⁺ 429.15

Step 3: Tert-butyl4-([6-[6-oxo-5-(trifluoromethyl)-1-[[2-(trimethylsilyl)ethoxy]methyl]-1,6-dihydropyridazin-4-yl]-5H,6H,7H-pyrrolo[3,4-b]pyridin-2-yl]oxy)piperidine-1-carboxylate

A solution of5-[2-hydroxy-5H,6H,7H-pyrrolo[3,4-b]pyridin-6-yl]-4-(trifluoromethyl)-2-[[2-(trimethylsilyl)ethoxy]methyl]-2,3-dihydropyridazin-3-one(200 mg, 0.47 mmol, 1.00 equiv), Ag₂CO₃ (247 mg, 2.00 equiv), andtert-butyl 4-iodopiperidine-1-carboxylate (416 mg, 1.34 mmol, 3.00equiv) in DMF (15 mL) was stirred for 4 h at 80° C. The resultingsolution was extracted with 3×10 mL of EtOAc and the organic layerscombined. After concentration, the residue was applied onto a silica gelcolumn with EtOAc/petroleum ether (1:9) to afford 150 mg (53%) of titlecompound as a yellow oil. LCMS: [M+H]⁺ 612.30.

Step 4:5-[2-(Piperidin-4-yloxy)-5H,6H,7H-pyrrolo[3,4-b]pyridin-6-yl]-4-(trifluoromethyl)-2,3-dihydropyridazin-3-one

A solution of tert-butyl4-([6-[6-oxo-5-(trifluoromethyl)-1-[[2-(trimethylsilyl)ethoxy]methyl]-1,6-dihydropyridazin-4-yl]-5H,6H,7H-pyrrolo[3,4-b]pyridin-2-yl]oxy)piperidine-1-carboxylate(150 mg, 0.25 mmol, 1.00 equiv) in HCl/dioxane (15 mL, 4M) was stirredovernight at 25° C. The pH value of the solution was adjusted to 8 withammonia (100%). The crude product was purified by Prep-HPLC to afford64.8 mg (69%) of title compound as a white solid. LCMS: [M+H]⁺ 382.15[M+H].

Step 5:5-[2-[(1-Acetylpiperidin-4-yl)oxy]-5H,6H,7H-pyrrolo[3,4-b]pyridin-6-yl]-4-(trifluoromethyl)-2,3-dihydropyridazin-3-one

A solution of5-[2-(piperidin-4-yloxy)-5H,6H,7H-pyrrolo[3,4-b]pyridin-6-yl]-4-(trifluoromethyl)-2,3-dihydropyridazin-3-one(300 mg, 0.79 mmol, 1.00 equiv), TEA (239 mg, 2.36 mmol, 3.00 equiv),and EtOAc (160 mg, 1.57 mmol, 2.00 equiv) in DCM (15 mL) was stirred for1 h at 25° C. The resulting solution was quenched by 20 mL of water andextracted with 3×15 mL of DCM and the organic layers combined. Afterconcentration, the crude product was purified by Flash-Prep-HPLC toafford 70.2 mg (21%) of title compound as a white solid. LCMS: [M+H]⁺424.15. ¹H NMR (400 MHz, Methanol-d₄) δ 8.07 (s, 1H), 7.69 (d, J=8.4 Hz,1H), 6.76 (d, J=8.4 Hz, 1H), 5.39-5.28 (m, 1H), 5.03 (s, 2H), 4.92 (s,2H), 3.97-3.89 (m, 1H), 3.86-3.75 (m, 1H), 3.57-3.44 (m, 2H), 2.15 (s,3H), 2.14-1.97 (m, 2H), 1.89-1.70 (m, 2H).

Example 9: In Vitro Assays Recombinant PARP Enzymes

A portion of TIPARP (residues 456 to 657 of NP_056323.2 (SEQ ID NO: 1),GenBank Accession No. NM_015508.4) was overexpressed in E. coli cells.An N-terminal fusion tag, MHHHHHHSSGVDLGTENLYFQSNAGLNDIFEAQKIEWHE (SEQID NO: 7), was used to purify the protein from cell lysates. The fusiontag was left on the protein for use in the probe displacement assay.

A portion of PARP10 (residues 808 to 1025 of NP_116178.2 (SEQ ID NO: 2),GenBank Accession No. NM_032789.4) was overexpressed in E. coli cells.An N-terminal fusion tag, MAHHHHHHENLYFQSM (SEQ ID NO: 8), was used topurify the protein from cell lysates. The fusion tag was left on theprotein for use in the probe displacement assay.

A portion of PARP12 (residues 489 to 684 of NP_073587.1 (SEQ ID NO: 3),GenBank Accession No. NM_022750.3) was overexpressed in Sf9 cells. AnN-terminal fusion tag, MAHHHHHHENLYFQSM (SEQ ID NO: 8), was used topurify the protein from cell lysates. The fusion tag was left on theprotein for use in the probe displacement assay.

A portion of PARP14 (residues 1611 to 1801 of NP_060024.2 (SEQ ID NO:4), GenBank Accession No. NM_017554) was overexpressed in E. coli cells.An N-terminal fusion tag, MHHHHHHSSGVDLGTENLYFQSNA (SEQ ID NO: 9), wasused to purify the protein from cell lysates. The fusion tag was left onthe protein for use in the probe displacement assay.

A portion of PARP15 (residues 481 to 678 of NP_689828.1 (SEQ ID NO: 5),GenBank Accession No. NM_152615) was overexpressed in f9 cells. AnN-terminal fusion tag, MAHHHHHHSSGVDLGTENLYFQSM (SEQ ID NO: 10), wasused to purify the protein from cell lysates. The fusion tag was left onthe protein for use in the probe displacement assay.

A portion of PARP16 (residues 5 to 279 of NP_060321.3 (SEQ ID NO: 6),GenBank Accession No. NM_017851) was overexpressed in E. coli cells. AnN-terminal fusion tag, MHHHHHHSSGVDLGTENLYFQSNA (SEQ ID NO: 9), was usedto purify the protein from cell lysates. The fusion tag was left on theprotein for use in the probe displacement assay.

In Vitro Probe Displacement Assay for Assessing Binding of Inhibitors toTIPARP, PARP10, PARP14 and PARP16

Displacement of a Probe C binding to monoPARP active sites was measuredusing a time-resolved fluorescence resonance energy transfer (TR-FRET)assay. 20 nL of a dose response curve of each test compound was spottedin black 384-well polystyrene proxiplates (Perkin Elmer) using aMosquito (TTP Labtech). Reactions were performed in an 8 μL volume byadding 6 μL of the monoPARP and Probe C in assay buffer (20 mM HEPESpH=8, 100 mM NaCl, 0.1% bovine serum albumin, 2 mM DTT and 0.002%Tween20), incubating with test compound at 25° C. for 30 min, thenadding 2 μL of ULight-anti 6×His and LANCE Eu-W1024 labeled streptavidin(Perkin Elmer). The final concentrations of monoPARP, Probe C,ULight-anti 6×His and LANCE Eu-W1024 labeled streptavidin are listed inTable 3. Binding reactions were equilibrated at 25° C. for an additional30 min, then read on an Envision platereader equipped with aLANCE/DELFIA top mirror (Perkin Elmer) using excitation of 320 nm andemission of 615 nm and 665 nM with a 90 μs delay. The ratio of the665/615 nm emission were calculated for each well to determine theamount of complex of monoPARP and Probe C in each well.

TABLE 3 Assay conditions for monoPARP probe displacement assays wherethe streptavidin is labeled with TR-FRET donor LANCE Eu- Enzyme Probe CULight-anti W1024 labeled Target (nM) (nM) 6xHis (nM) streptavidin (nM)TIPARP 6 2 4 0.25 PARP10 6 0.5 2 0.25 PARP14 6 2 10 0.25 PARP16 3 1 60.25

In Vitro Probe Displacement Assay for Assessing Binding of Inhibitors toPARP12 and PARP15

Displacement of a Probe C binding to monoPARP active sites was measuredusing a time-resolved fluorescence resonance energy transfer (TR-FRET)assay. 20 nL of a dose response curve of each test compound was spottedin black 384-well polystyrene proxiplates (Perkin Elmer) using aMosquito (TTP Labtech). Reactions were performed in a 8 μL volume byadding 6 μL of the monoPARP and Probe C in assay buffer (20 mM HEPESpH=8, 100 mM NaCl, 0.1% bovine serum albumin, 2 mM DTT and 0.002%Tween20), incubating with test compound at 25° C. for 30 min, thenadding 2 μL of ULight-streptavidin and LANCE Eu-W1024 Anti-6×His (PerkinElmer). The final concentrations of monoPARP, Probe C,ULight-streptavidin and LANCE Eu-W1024 Anti-6×His are listed in Table 4.Binding reactions were equilibrated at 25° C. for an additional 30 min,then read on an Envision platereader equipped with a LANCE/DELFIA topmirror (Perkin Elmer) using excitation of 320 nm and emission of 615 nmand 665 nM with a 90 μs delay. The ratio of the 665/615 nm emission werecalculated for each well to determine the amount of complex of monoPARPand Probe C in each well.

TABLE 4 Assay conditions for monoPARP probe displacement assays wherethe anti-His antibody is labeled with TR-FRET donor LANCE Eu- EnzymeProbe C ULight- labeled W1024 anti Target (nM) (nM) streptavidin (nM)6xHis (nM) PARP12 6 32 10 1.25 PARP15 1.5 8 2 0.5

Data Analysis for all In Vitro Assays

Control wells containing a negative control of 0.25% DMSO vehicle or apositive control of 100 μM Compound A were used to calculate the %inhibition as described below:

${\%\mspace{14mu}{inhibition}} = {100 \times \frac{{TRF}_{cmpd} - {TRF}_{\min}}{{TRF}_{\max} - {TRF}_{\min}}}$

where TRF_(cmpd) is the TR-FRET ratio from the compound treated well,TRF_(min) is the TR-FRET ratio from the Compound A-treated positivecontrol well and TRF_(max) is the TR-FRET ratio from the DMSO-treatednegative control well.

The % inhibition values were plotted as a function of compoundconcentration and the following 4-parameter fit was applied to derivethe IC₅₀ values:

$Y = {{Bottom} + \frac{\left( {{Top} - {Bottom}} \right)}{\left( {1 + \left( \frac{X}{{IC}_{50}} \right)^{{Hill}\mspace{14mu}{Coefficient}}} \right.}}$

where top and bottom are normally allowed to float, but may be fixed at100 or 0 respectively in a 3-parameter fit. The Hill Coefficient isnormally allowed to float but may also be fixed at 1 in a 3-parameterfit. Y is the % inhibition and X is the compound concentration.

Validation of In Vitro Probe Displacement Assays

The probe displacement assays were validated by outcompeting Probe Cwith Compound A, an analog that does not contain a linker or biotingroup. The assays for TIPARP, PARP10, PARP12, PARP14, PARP15 and PARP16were set up as described above, and the results are shown in FIGS.4A-4F.

FIGS. 4A-4F are validation results of the in vitro probe displacementbinding assays. Dose response curves for Compound A were generated usingeach assay to confirm that Probe C was able to be outcompeted from themonoPARP enzyme. IC₅₀ values were TIPARP=7 nM, PARP10=80 nM, PARP12=200nM, PARP14=50 nM, PARP15=60 nM and PARP16=100 nM.

Example 10: Live Cell Assays NanoLuc Plasmids

MonoPARP genes from Table 5 were cloned into the pcDNA3.1-mammalianexpression vector as a NanoLuc fusion with NanoLuc on the N- orC-terminus as indicated. The sequence of the NanoLuc tag is as follows:

NanoLuc amino acid sequence (SEQ ID NO: 11):MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGS LLFRVTINGVTGWRLCERILANanoLuc nucleic acid sequence (SEQ ID NO: 12):atggtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggctgtgcga acgcattctggcgtaa

TABLE 5 Details for the monoPARP-NanoLuc fusion genes used Genbank AminoAcid NanoLuc Accession Plasmid Name Number Residues Position Full-lengthTIPARP NM_015508 1-657 of C-terminus NP_056323.2 (SEQ ID NO: 1)Catalytic domain NM_015508 456-657 of C-terminus TIPARP NP_056323.2 (SEQID NO: 1) PARP10 NM_032789 808-1025 of C-terminus NP_116178.2 (SEQ IDNO: 2) PARP12 NM_022750.3 489-684 of C-terminus NP_073587.1 (SEQ ID NO:3) PARP14 NM_017554 1611-1801 of N-terminus NP_060024.2 (SEQ ID NO: 4)

NanoBRET Probe Displacement Assay for Assessing Binding of Inhibitors toTIPARP, PARP10, PARP12 and PARP14

Displacement of a fluorescently-labeled compound Probe A binding toNanoLuc-tagged monoPARP enzymes was measured in live cells using abioluminescence resonance energy transfer (NanoBRET) assay. TIPARP,PARP10, PARP12 or PARP14 fused to a NanoLuc tag were overexpressed in293T cells (ATCC) using the plasmids described herein. Plasmid DNA andempty vector DNA were added to phenol red free OptiMEM (Thermo Fisher)as shown in Table 6 in a total volume of 2.456 mL. 157 μL of Fugene HD(Promega) was added to the DNA mixture and allowed to incubate 5 min at25° C.

TABLE 6 Concentration of Concentration of Plasmid Empty Vector Probe APlasmid Name (μg/mL) (μg/mL) (nM) Full-length TIPARP 0.2 19.8 9Catalytic domain 0.2 19.8 80 TIPARP PARP10 2 18 7 PARP12 2 18 300 PARP140.2 19.8 100

Next, 2.375 mL of the plasmid-Fugene mixture were added to 20 million293T cells in DMEM (Thermo Fisher) supplemented with 10% FBS (VWR). Thetransfection was incubated for 24 h at 37° C. in an incubator containingair supplemented with 5% CO₂. The cells were resuspended in phenol redfree OptiMEM media. Transfected 293T cells were diluted to 500,000 cellsper mL and Probe A was added to a final concentration as shown in Table6. 40 μL of cells were then added to white polystyrene 384-wellnon-binding surface microplate (Corning). 40 nL of a dose response curvediluted in DMSO of each test compound was added to the cell plate usinga Mosquito (TTP Labtech) and the plate was incubated at 37° C. in anincubator containing air supplemented with 5% CO₂ for 2 h. The assayplate was allowed to equilibrate to room temperature (25° C.), then 20μL per well of NanoBRET substrate (Promega) was added to the plate(1:166 dilution of NanoBRET substrate, 1:500 dilution of NanoLucextracellular inhibitor in OptiMEM without phenol red). Filteredluminescence was measured on an Envision (Perkin Elmer) equipped with adual 585 nm mirror, 460±40 nm bandpass filter (donor) and 610±50 nmlongpass filter (acceptor).

TABLE 7 Positive control Final Concentration Assay compound (μM)Full-length TIPARP Compound B 0.1 Catalytic domain TIPARP Compound B 0.2PARP10 Compound C 2 PARP12 Compound B 1 PARP14 Compound D 5

Data Analysis for NanoBRET Assays

BRET ratio was measured as shown below:

${{BRET}\mspace{14mu}{ratio}} = \frac{{Emission}\mspace{14mu}{at}\mspace{14mu} 610\mspace{14mu}{nm}}{Luminescence}$

Control wells containing a negative control of 0.2% DMSO vehicle or apositive control were used to calculate the % inhibition as describedbelow:

${\%\mspace{14mu}{inhibition}} = {100 \times \frac{{{BRET}\mspace{14mu}{ratio}_{cmpd}} - {{BRET}\mspace{14mu}{ratio}_{\min}}}{{{BRET}\mspace{14mu}{ratio}_{\max}} - {{BRET}\mspace{14mu}{ratio}_{\min}}}}$

where BRET ratio_(cmpd) is the BRET ratio from the compound treatedwell, BRET ratio_(min) is the BRET ratio from the positive control wellsand BRET ratio_(max) is the BRET ratio from the DMSO treated negativecontrol well.

The % inhibition values were plotted as a function of compoundconcentration and the following 4-parameter fit was applied to derivethe IC₅₀ values:

$Y = {{Bottom} + \frac{\left( {{Top} - {Bottom}} \right)}{\left( {1 + \left( \frac{X}{{IC}_{50}} \right)^{{Hill}\mspace{14mu}{Coefficient}}} \right.}}$

where top and bottom are normally allowed to float, but may be fixed at100 or 0 respectively in a 3-parameter fit. The Hill Coefficient isnormally allowed to float but may also be fixed at 1 in a 3-parameterfit. Y is the % inhibition and X is the compound concentration.

Validation of NanoBRET Probe Displacement Assays

The probe displacement assays were validated by outcompeting Probe Awith Compound A in the PARP14 NanoBRET assay, Compound B in the TIPARPNanoBRET assay, Compound C in the PARP10 NanoBRET assay and Compound Bin the PARP12 NanoBRET assay. The compounds used to test the probedisplacement of Probe A are analogs that do not contain a linker orfluorescent tag. The assays for TIPARP full-length and catalytic domain,PARP10, PARP12 and PARP14 were set up as described above, and theresults are shown in FIG. 5.

FIGS. 5A-5D are validation results of the NanoBRET probe displacementbinding assays. Dose response curves for control compounds weregenerated using each assay to confirm that Probe A was able to beoutcompeted from the monoPARP enzyme. IC₅₀ values were full-lengthTIPARP Compound B=4 nM, catalytic domain TIPARP Compound B=7 nM, PARP10Compound C=4 nM, PARP12 Compound B=170 nM, PARP14 compound A=30 nM.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

REFERENCES

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What is claimed is:
 1. A method of identifying an inhibitor for Poly(ADP-ribose) polymerase (PARP), the method comprising: combining (i) apolypeptide comprising a PARP catalytic domain wherein the polypeptideis labeled with a donor fluorophore, (ii) a PARP probe, wherein the PARPprobe is labeled with an acceptor fluorophore, and (iii) a testcompound; exposing the donor fluorophore to excitation light; measuringa signal produced by the acceptor fluorophore; and identifying the testcompound as an inhibitor for PARP based on the signal produced by theacceptor fluorophore.
 2. The method of claim 1, wherein the PARP probeis a compound having the structure:

or a salt thereof.
 3. The method of claim 1, further comprisingidentifying the test compound as an inhibitor for PARP if the signalproduced by the acceptor fluorophore is decreased as compared to areference level.
 4. The method of claim 3, wherein the reference levelis the signal produced by the acceptor fluorophore in the absence of thetest compound.
 5. The method of claim 1, wherein the PARP is PARP1,PARP2, PARP3, PARP4, PARP5a, PARP5b, PARP6, TIPARP, PARP8, PARP9,PARP10, PARP11, PARP12, PARP13, PARP14, PARP15, or PARP16.
 6. The methodof claim 1, wherein the PARP probe is biotinylated, and the acceptorfluorophore is attached to streptavidin.
 7. The method of claim 1,wherein the polypeptide comprises a polyhistidine tag.
 8. The method ofclaim 1, wherein the test compound is a small molecule, a nucleic acid,a polypeptide, or an antibody or antigen-binding fragment thereof.
 9. Amethod of identifying an inhibitor for Poly (ADP-ribose) polymerase(PARP), the method comprising: combining (i) a polypeptide comprising aPARP catalytic domain, wherein the polypeptide is labeled with anacceptor fluorophore; (ii) a PARP probe, wherein the PARP probe islabeled with a donor fluorophore, and (iii) a test compound; exposingthe donor fluorophore to excitation light; measuring a signal producedby the acceptor fluorophore in the presence of a test compound; andidentifying the test compound as an inhibitor for PARP based on thesignal produced by the acceptor fluorophore.
 10. The method of claim 9,wherein the PARP probe is a compound having the structure:

or a salt thereof.
 11. The method of claim 9, further comprisingidentifying the test compound as an inhibitor for PARP if the signalproduced by the acceptor fluorophore is decreased as compared to areference level.
 12. The method of claim 11, wherein the reference levelis the signal produced by the acceptor fluorophore in the absence of thetest compound.
 13. The method of claim 9, wherein the PARP is PARP1,PARP2, PARP3, PARP4 PARP5a, PARP5, PARP6, TIPARP, PARP8, PARP9, PARP10,PARP11, PARP12, PARP13, PARP14, PARP15, or PARP16.
 14. The method ofclaim 9, wherein the test compound is a small molecule, a nucleic acid,a polypeptide, or an antibody or antigen-binding fragment thereof.
 15. Amethod of identifying an inhibitor for Poly (ADP-ribose) polymerase(PARP), the method comprising: contacting a polypeptide comprising aPARP catalytic domain with a PARP probe in the presence of a testcompound, wherein the PARP probe comprises a fluorophore; exposing theprobe to polarized excitation light, thereby generating fluorescence;determining a fluorescence polarization value of the fluorescence; andidentifying the test compound as an inhibitor for PARP based on thefluorescence polarization value of the fluorescence.
 16. The method ofclaim 15, further comprising identifying the test compound as aninhibitor for PARP if the fluorescence polarization value of thefluorescence is decreased as compared to a reference level.
 17. Themethod of claim 16, wherein the reference value is the fluorescencepolarization value of the fluorescence in the absence of the testcompound.
 18. The method of claim 15, wherein the PARP probe is acompound having the structure:

or or a salt thereof.
 19. The method of claim 15, wherein the PARP isPARP1, PARP2, PARP3, PARP4, PARP5a, PARP5b, PARP6, TIPARP, PARP8,PARP10, PARP1l, PARP12, PARP13, PARP14, PARP15, or PARP16.
 20. Themethod of claim 15, wherein the test compound is a small molecule, anucleic acid, a polypeptide, or an antibody or antigen-binding fragmentthereof.
 21. A method of identifying an inhibitor for Poly (ADP-ribose)polymerase (PARP), the method comprising: contacting a fusionpolypeptide with a PARP probe that comprises a fluorophore, wherein thefusion polypeptide comprises a PARP catalytic domain and a luciferaseenzyme; contacting the luciferase enzyme with a substrate to producelight, wherein the light can excite the fluorophore; measuring a signalproduced by the fluorophore in the presence of a test compound; andidentifying the test compound as an inhibitor for PARP based on thesignal produced by the fluorophore.
 22. The method of claim 21, furthercomprising identifying the test compound as an inhibitor for PARP if thesignal produced by the fluorophore is decreased as compared to areference level.
 23. The method of claim 22, wherein the reference levelis the signal produced by the fluorophore in the absence of the testcompound.
 24. The method of claim 21, wherein the PARP probe is acompound having the structure:

or a salt thereof.
 25. The method of claim 21, wherein the fusionpolypeptide comprises a sequence that is at least 85%, 90% or 95%identical to SEQ ID NO:
 11. 26. The method of claim 22, wherein the PARPis PARP1, PARP2, PARP4, PARP5a, PARP5b, PARP3, PARP6, TIPARP, PARP8,PARP9, PARP10, PARP11, PARP12, PARP13, PARP14, PARP15, or PARP16. 27.The method of claim 22, wherein the test compound is a small molecule, anucleic acid, a polypeptide, or an antibody or antigen-binding fragmentthereof.
 28. The method of claim 1, wherein the polypeptide comprising aPARP catalytic domain comprises a sequence that is at least 85%, 90% or95% identical to amino acid residues of 456 to 657 of NP_056323.2 (SEQID NO: 1).
 29. The method of claim 1, wherein the polypeptide comprisinga PARP catalytic domain comprises a sequence that is at least 85%, 90%or 95% identical to amino acid residues of 808 to 1025 of NP_116178.2(SEQ ID NO: 2).
 30. The method of claim 1, wherein the polypeptidecomprising a PARP catalytic domain comprises a sequence that is at least85%, 90% or 95% identical to amino acid residues of 489 to 684 ofNP_073587.1 (SEQ ID NO: 3).
 31. The method of claim 1, wherein thepolypeptide comprising a PARP catalytic domain comprises a sequence thatis at least 85%, 90% or 95% identical to amino acid residues of 1611 to1801 of NP_060024.2 (SEQ ID NO: 4).
 32. The method of claim 1, whereinthe polypeptide comprising a PARP catalytic domain comprises a sequencethat is at least 85%, 90% or 95% identical to amino acid residues of 481to 678 of NP_689828.1 (SEQ ID NO: 5).
 33. The method of claim 1, whereinthe polypeptide comprising a PARP catalytic domain comprises a sequencethat is at least 85%, 90% or 95% identical to amino acid residues of 5to 279 of NP_060321.3 (SEQ ID NO: 6).
 34. A PARP probe having astructure according to Formula (I):

or a salt thereof, wherein: L is a linking group having 5-30 spaceratoms selected from C, N, O, and S connecting the N atom of thepiperidinyl group of Formula (I) with group A; A is a fluorophore or anaffinity tag.
 35. The PARP probe of claim 34 wherein L is

a is 0, 1, or 2; b is 1-26; and c is 0, 1, or 2; wherein the sum ofa+b+c is 1 to 26, or L is a chain of 5-30 atoms in length comprising—(CH₂CH₂O)_(d)— wherein d is 2-10.
 36. The PARP probe of claim 34wherein A is:

or a salt thereof.
 37. The PARP probe of claim 34 which is a compoundhaving the structure:

or a salt thereof.